Use of fluorinated derivatives of 4-aminopyridine in therapeutics and medical imaging

ABSTRACT

The present disclosure provides novel compounds, including compounds that bind to potassium channels, methods for their manufacture, and methods for their use, including their use to diagnose and/or assess traumatic brain injury and use to treat dymeylinating diseases, and/or in vivo imaging of the central neverous system, and to diagnose and/or assess the progression of MS or other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is is a continuation of U.S. patent application Ser.No. 16/584,071 filed Sep. 26, 2019, which is a continuation of U.S.patent application Ser. No. 15/452,179 filed Mar. 7, 2017, which is acontinuation of U.S. patent application Ser. No. 14/329,597 filed Jul.11, 2014, which is a continuation-in-part of U.S. patent applicationSer. No. 13/897,035 filed May 17, 2013 and PCT ApplicationPCT/US2013/041638 filed May 17, 2013, both of which claim priority toU.S. Provisional Patent Application Ser. No. 61/648,214 filed May 17,2012. U.S. patent application Ser. No. 14/329,597 also claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.61/845,878 filed Jul. 12, 2013. The entire contents of each of theabove-referenced disclosures are specifically incorporated herein byreference without disclaimer.

BACKGROUND OF THE INVENTION I. Field of the Invention

The present invention relates generally to the fields of biology,chemistry and medicine. More particularly, it concerns derivatives ofpotassium channel inhibitors, including derivatives of 4-aminopyridine,and methods of making and using thereof, including for the treatment andmedical imaging of neurodegenerative conditions.

II. Description of Related Art

With nearly 400,000 people affected in the U.S. and 2.5 millionworldwide, Multiple Sclerosis (MS) is the most common neurodegenerativecondition in young adults (Calabresi, 2007). The progressivedemyelination of neurons in the brain leads to diverse neurologicalsymptoms. Myelin is the multilayered membrane that surrounds most axonsof the central and peripheral nervous systems and is essential for thepropagation of rapid nerve impulses. In people with MS, the myelinsheath that normal covers the axons is lost and this leads to aberrantleakage of potassium ions from the axon and improper impulse conduction.

One approach to treat MS or to mitigate the symptoms associated with MSis to block potassium channels to reduce the leakage of potassium ions,thus enhancing impulse conduction. In January 2010, the FDA approved4-aminopyridine (4-AP), as a therapy for MS (Ampyra, AcordaTherapeutics, Inc., 2010). 4-AP is a relatively selective blocker ofK_(v)1 family of K⁺ channels (Wulff et al., 2009). By blocking K⁺channels, impulse conduction along the axon is partially restored andsymptoms ameliorate.

To develop new neuroprotective therapies for MS or otherneurodegenerative diseases, it is essential to have proper tools todiagnose and assess disease progression.

According to CDC around 1.74 million people sustain a traumatic braininjury in the U.S. each year. Most of these injuries are mild (75%) andcertain populations are at a higher risk: men aged 0-4, 15-19 and over60 as well as military personnel and people engaged in contact sports.Recent studies have shown that even mild TBIs can have seriousconsequences later in life. TBI has been linked to depression, anxiety,substance abuse and suicide. All these reasons make screening for TBIparticularly important.

Currently, the diagnosis of TBI is based on clinical evaluation aided byComputed Tomography (CT) or Magnetic Resonance Diffusion Tensor Imaging(MR-DTI). CT scans are very useful for detecting mass lesions andfractures but do not allow visualization of mild TBIs. More recently,MR-DTI has emerged as a sensitive method to evaluate white matterintegrity in TBI but it too can be difficult to interpret.

During TBI, compression or stretching of the brain often causes damageto axons and/or the myelin sheath. Oligodendrocytes, the cellsresponsible for producing and maintaining myelin, have also been shownto be sensitive to TBI (Flygt et al., 2013; Sharp and Ham, 2011; Moreyet al., 2012). Oligodendrocyte injury results in axonal demyelination.In addition to facilitating raping nerve conduaction velocities, myelinprovides axonal protection, such that demyelinated axons are prone todegeneration. Therefore, TBI-induced damage to myelin and/oroligodendrocytes likely contributes to the acute and long-term clinicalmanifestations of TBI.

Axonal proteins are compartmentalized in myelinated axons, with thevoltage-gated sodium channels concentrated at the unmyelinated node ofRanvier and the rectifying potassium (K+) channels residing under themyelin sheath (Waxman and Ritchie, 1993). Following demyelination, likethat which occurs in multiple sclerosis (MS) and TBI, the K+ channelsbecome exposed and leaky. In 2010, the FDA approved 4-amino-pyridine(4-AP, Ampyra®) as a drug to improve symptoms in people with MS. 4-AP isa K+ channel blocker that binds to the exposed channels on demyelinatedaxons, which reduces the aberrant efflux of K+ ions and enhancesneuronal conduction. Since 4-AP selectively targets K+ channels thathave become uncovered as a result of demyelination we propose to testits usefulness as a tracer for demyelinated axons.

Not much is known about the role of axonal K+ channels and the effectsof 4-AP in TBI. However, there have been numerous studies looking at theeffects of 4-AP after Spinal Cord Injury (SCI; Blight et al., 1989;Blight et al., 1991; Hayes et al., 1993; Fehlings and Nashmi, 1996;Gruner and Yee, 1999). Similarly to TBI, SCI is an injury to the CNSthat occurs after a violent impact. Depending on the location andseverity of the injury the symptoms can vary from partial loss ofmovement and sensation (incomplete injury) to complete loss. In cases ofincomplete injury, 4-AP has been shown to enhance neuronal conductionthrough injured areas both in animals and in humans (Blight et al.,1989; Blight et al., 1991; Hayes et al., 1993). In addition, injuredspinal cord areas have been shown to have higher pharmacologicalsensitivity to 4-AP (Fehlings and Nashmi, 1996), which agrees with ourhypothesis that K+ channels on demyelinated fibers are more accessibleand suggest the potential of using radioactive 4-AP to map injuredareas. The similarities between TBI and SCI in etiology and at thehistopathological level justify evaluating 4-AP based PET tracers forTBI. In addition, if 4-AP is found to localize to injured areas in TBIit could also be useful for restoring function/ameliorating symptoms inTBI patients. Fluorine-18 is the preferred isotope for PET imagingbecause its long half-life allows for off-site production andcommercialization. In addition, its low positron energy gives higherresolution than for example carbon-11. We have also shown that thesefluorinated molecules have very similar properties to 4-AP both in vitroand in vivo indicating that fluorination does not disrupt its propertiesand therefore these molecules could be used as surrogates of 4-AP.

Thus, there is a pressing need for new, accurate methods to evaluate anddiagnose TBI.

SUMMARY OF THE INVENTION

In some embodiments, there are provided compounds that bind to potassiumchannels, methods for their manufacture, and methods for their use. In aparticular embodiment, the compounds may be compounds of formula (I):

wherein R₁, R₂, R₃, and R₄ are each independently selected from thegroup consisting of H, (CH₂)_(n)X, CH₂OCH₂CH₂X, NH₂, CH₂H, CF₃, OCH₃,OCH₂F, OCHF₂, OCF₃ and R₅ is selected from the group consisting of H,(CH₂)_(m)X, OH, COOCF₃, COOC(CH₃)₃, and COO(CH₂)_(m)X; wherein n=0, 1,2, 3, 4, or 5 and m=2, 3, 4, or 5; wherein X represents a fluorine atomor an isotope thereof; as well as pharmaceutically acceptable salts,tautomers, or deuterated versions thereof.

In one aspect, the isotope of fluorine is a radioactive isotope. In aparticular aspect, the fluorine isotope is ¹⁸F. In some aspects, any ofC, N, O is optionally replaced by an isotope thereof. An isotope of C,N, O may be any known C, N, O isotope. In particular aspects, theisotope is a radioisotope. For example, any of C, N, O may be optionallyreplaced by the isotope ¹¹C, ¹³N, ¹⁵O, respectively.

In further embodiments, at least one of R₁, R₂, R₃, R₄ and R₅ is nothydrogen. In still further embodiments, at least one of R₁, R₂, R₃, R₄and R₅ contains a fluorine atom or an isotope thereof. In certainaspects, when R₂ is NH₂, CH₂OH, a nonradioactive fluorine, or CF₃, atleast one of R₁, R₃, R₄, and R₅ is not hydrogen. In additional aspects,when R₄ is NH₂, CH₂OH, a nonradioactive fluorine, or CF₃, at least oneof R₁, R₂, R₄, and R₅ is not hydrogen.

In some embodiments, the compounds are not the following compounds:

In some embodiments, the compounds have the following formulas:

In certain embodiments, there are provided compounds of formula (II):

wherein M is (CH₂)_(n)Y and wherein n=0, 1, or 2, and Y is fluorine oran isotope thereof, as well as pharmaceutical acceptable salts,tautomers, or deuterated versions thereof.

In certain embodiments, M is CH₂F, or (CH₂)₂F. In further embodiments, Mis ¹⁸F, CH₂ ¹⁸F, or (CH₂)₂ ¹⁸F. In some aspects, any of C, N, O isoptionally replaced by an isotope thereof. An isotope of C, N, O may beany known C, N, O isotope. In particular aspects, the isotope is aradioisotope. For example, any of C, N, O may be optionally replaced bythe isotope ¹¹C, ¹³N, ¹⁵O, respectively.

In certain embodiments, there are provided compounds of formula (III):

wherein R is selected from the group consisting of CH₃, CH₂F, CHF₂, andCF₃, and wherein C is substituted by ¹¹C or at least one of F issubstituted by ¹⁸F in R. For instance, R is ¹¹CH₃, CH₂ ¹⁸F, CHF¹⁸F,CH(¹⁸F)₂, C¹⁸FF₂, C(¹⁸F)₂F, or C(¹⁸F)₃.

Certain embodiments are directed to the compounds of formula (IV):

wherein R is selected from the group consisting of CF₃, CH₂F, CH₃CH₂F,C(CH₃)₃, and wherein at least one of F or H in the R group issubstituted by ¹⁸F. Non-limiting examples include CH₂ ¹⁸F, CHF¹⁸F,CH(¹⁸F)₂, C¹⁸FF₂, C(¹⁸F)₂F, C(¹⁸F)₃, CH₃CH₂ ¹⁸F, and C(CH₃)₂ ¹⁸F.

In some aspects, any of C, N, O in the compounds described herein isoptionally replaced by an isotope thereof. An isotope of C, N, O may beany known C, N, O isotope. In particular aspects, the isotope is aradioisotope. For example, any of C, N, O in the compounds of formula(I)-(IV) may be optionally replaced by the isotope ¹¹C, ¹³N, ¹⁵O,respectively.

In some embodiments there are provided pharmaceutical compositionscomprising one or more of the above compounds and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositionsfurther comprise one or more pharmaceutically acceptable excipients. Insome embodiments, the composition is formulated for controlled releaseof any of the compounds disclosed herein.

Certain embodiments are directed to a kit comprising one or more of theabove compounds. In further aspects, there are provided a kit comprisingone or more of the above compounds comprising a radioisotope.

In some embodiments, there are provided imaging methods comprisingadministering to a subject in need thereof the imaging agent describedherein and detecting the compound comprised in the imaging agent in thesubject. In some aspects, the amount of the compound in the subject isquantified. In further aspects, a demyelinated region in an axon in thesubject is detected via a detection of the compound in the subject. Instill further aspects, the compound administered to the subject mayblock potassium channels located at the demyelinated region in an axonin the subject.

In certain embodiments, the imaging is effected by a radiodiagnosticmethod. The radiodiagnostic method may be performed by any instrumentcapable of detecting radiation by the compounds. Exemplaryradiodiagnostic methods include, but not limited to, Positron EmissionTomography (PET), PET-Time-Activity Curve (TAC) or PET-MagneticResonance Imaging (MRI). In particular aspect, the radiodiagnosticmethod is PET.

Certain embodiments are directed to an imaging agent comprising acompound described herein wherein the compound contains an isotope. Insome embodiments, the isotopes are isotopes of F, O, N and C. Inparticular aspects, the isotope is a fluorine isotope. In furtherembodiments, the isotope is a radioisotope. In still furtherembodiments, the radioisotope is ¹⁸F, ¹⁵O, ¹³N or ¹¹C. In particularembodiments, the isotope is ¹⁸F. For example, an imaging agent maycomprise a derivative of 4-AP, including, but not limited to,[¹⁸F]-3-fluoro-4-aminopyridine, [¹⁸F]-3-fluoro-methyl-4-aminopyridine,and [¹⁸F]-3-fluoro-ethyl-4-aminopyridine.

In some embodiments, there are provided methods the use of novelcompounds as described herein, including for the treatment and/or invivo imaging of the central nervous system to diagnose and/or assess theprogression of MS or other diseases.

In some embodiments, there are provided methods for diagnosing traumaticbrain injury (TBI) or evaluating the progression of TBI comprisingadministering to a subject in need thereof an imaging agent describedherein and detecting the compound comprised in the imaging agent in thesubject.

In some embodiments, there are provided methods of treating ademyelinating disease or mitigating a symptom of a demyelinating diseasecomprising administering to a subject in need thereof an effectiveamount of a compound as defined above.

In further embodiments, there are provided methods of treating TBI ormitigating a symptom of TBI comprising administering to a subject inneed thereof an effective amount of a compound as defined above.

It is specifically contemplated that in certain embodiments, methodsrelated to therapy and/diagnostics involve a subject that is a humanpatient.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

“Effective amount” or “therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease. In someembodiments, the subject is administered at least about 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 mg/kg (or any range derivable therein).

The amount of the compound that is administered or taken by the patientmay be based on the patient's weight (in kilograms). Therefore, in someembodiments, the patient is administered or takes a dose or multipledoses amounting to about, at least about, or at most about 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5,12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5,18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320,325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390,395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490,500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610,620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725,730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840,850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960,970, 975, 980, 990, 1000 micrograms/kilogram (kg) or mg/kg, or any rangederivable therein. In some aspects, the pharmaceutically effectiveamount comprises a dose from about 0.0001 mg/kg/day to about 100mg/kg/day. In further aspects, the effective amount comprises a dosefrom about 0.01 mg/kg/day to about 5 mg/kg/day. In still furtheraspects, the dose is about 0.25 mg/kg/day.

The composition may be administered to (or taken by) the patient 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moretimes, or any range derivable therein, and they may be administeredevery 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or any rangederivable therein. It is specifically contemplated that the compositionmay be administered once daily, twice daily, three times daily, fourtimes daily, five times daily, or six times daily (or any rangederivable therein) and/or as needed to the patient. Alternatively, thecomposition may be administered every 2, 4, 6, 8, 12 or 24 hours (or anyrange derivable therein) to or by the patient.

In some embodiments, the compounds described herein are comprised in apharmaceutical composition. In further embodiments, the compoundsdescribed herein and optional one or more additional active agents, canbe optionally combined with one or more pharmaceutically acceptableexcipients and formulated for administration via epidural,introperitoneal, intramuscular, cutaneous, subcutaneous or intravenousinjection. In some aspects, the compounds or the composition isadministered by aerosol, infusion, or topical, nasal, oral, anal,ocular, or otic delivery. In further embodiments, the pharmaceuticalcomposition is formulated for controlled release.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylicacids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

In certain embodiments, the demyelinating disease includes, but is notlimited to, multiple sclerosis, spinal cord compression, ischemia, acutedisseminated encephalomyelitis, optic neuromyelitis, leukodystrophy,progressive multifocal leukoencephalopathy, metabolic disorders, toxicexposure, congenital demylinating disease, peripheral neuropathy,encephalomyelitis, central pontine myelolysis, Anti-MAG Disease,Guillain-Barre syndrome, chronic inflammatory demyelinatingpolyneuropathy, or multifocal motor neuropathy (MMN). In particularembodiments, the demyelinating disease is multiple sclerosis.

In additional embodiments, leukodystrophy includes, but is not limitedto, adrenoleukodystrophy, Alexander's Disease, Canavan Disease, KrabbeDisease, Metachromatic Leukodystrophy, Pelizaeus-Merzbacher Disease,vanishing white matter disease, Refsum Disease, Cockayne Syndrome, Vander Knapp Syndrome, or Zellweger Syndrome.

In some embodiments, there are provided methods for diagnosing ademyelinating disease or evaluating the progression of a demyelinatingdisease comprising administering to a subject in need thereof theimaging agent described herein and detecting the compound comprised inthe imaging agent in the subject. In certain aspects, the compound isdetected by a radiodiagnostic method, including, but not limited to PET,TAC, or PET-MRI. In particular aspects, the compound is detected by PET.

In some aspects, the subject is a mammal. In particular aspects, thesubject is a human. In additional aspects, the subject is a healthyindividual. In further aspects, the subject is a verified or putativeanimal model of myelin-associated neuropathy. For example, in someembodiments, the animal model is DTA model, cuprizone-induceddemyelination model, a lysolecithin injection model or experimentalautoimmune encephalomyelitis (EAE) model. In still further aspects, theanimal model is a mouse mutant with altered nodal environ, including,but not limited to, shiverer, trembler, jimpy, P0 null, E-cadherin null,Mag null, Dystrophic laminin α2, Cgt null, Contactin null, Caspr null,Cst null, Caspr2 null, Tag1 null, Dystroglycan, quivering Spectrin βIV,Nrcam null, and Na+ channel 2 null.

In some embodiments, the subject is at risk for traumatic brain injuryor a concussion. In some embodiments, the subject has a concussion orhas symptoms of a concussion. In some embodiments, the subject is anathlete or participates in athletic activities such as football, hockey,soccer, lacrosse, rugby, field hockey, horseback riding, bull riding,cheerleading, gymnastics, motocross, boxing, wrestling, base jumping,mountaineering, mixed martial arts, parkour, sky diving, free climing,skateboarding, surfing, luge, cliff diving, snowboarding, skiing, polevault, martial arts, cycling, racing, mountain biking, skating, cricket,basketball, roller derby, softball, baseball, polo, water polo, or otheractivities.

In some embodiments, there are provided methods for synthesizing thecompounds described herein. For example, 3-fluoromethyl-4-aminopyridineor 3-fluoroethyl-4-aminopyridine is produced by a method comprising (a)protecting the amino group of 4-aminopyridine-3-methanol or4-aminopyridine-3-ethanol with a protection group to form a firstintermediate compound, (b) fluorinating the first intermediate compoundby using a fluoro-containing reagent to form a second intermediatecompound, and (c) removing the protection group from the secondintermediate compound to form 3-fluoromethyl-4-aminopyridine or3-fluoroethyl-4-aminopyridine. In certain aspects, the protection groupis Boc (N-tert-butoxycarbonyl). In further aspects, thefluoro-containing reagent is XtalFluor E((Diethylamino)difluorosulfonium tetrafluoroborate).

In additional aspects, 3-fluoroethyl-4-aminopyridine may be synthesizedby a method comprising (a) converting 4-(Boc-amino)pyridine to4-(Boc-amino)pyridine-3-ethanol, (b) fluorinating4-(Boc-amino)pyridine-3-ethanol by using Xtal-Fluor, and c) removing Bocto form 3-fluoroethyl-4-aminopyridine.

Methods for producing the fluorine isotope containing compounds describeherein are also contemplated. For example,[¹⁸F]-3-fluoro-4-aminopyridine is produced by a method comprising (a)converting a compound having the structure A (4-(Boc-amino)pyridine) toan intermediate compound with structure B, and (b) fluorinating theintermediate structure B to form [¹⁸F]-3-fluoro-4-aminopyridine. A[¹⁸F]-containing reagent is supplied in the fluorination step.

A method for producing [¹⁸F]-3-fluoromethyl-4-aminopyridine or[¹⁸F]-3-fluoroethyl-4-aminopyridine is also provided. The methodcomprises (a) protecting the amino group of 4-aminopyridine-3-methanolor 4-aminopyridine-3-ethanol with a protection group to form a firstintermediate compound, (b) fluorinating the first intermediate compoundby using a [¹⁸F]-containing reagent to form a second intermediatecompound, and (c) removing the protection group from the secondintermediate compound to form [¹⁸F]-3-fluoromethyl-4-aminopyridine or[¹⁸F]-3-fluoroethyl-4 aminopyridine. In particular aspects, theprotection group is Boc (N-tert-butoxycarbonyl).

In additional aspects, [¹⁸F]-3-fluoroethyl-4-aminopyridine may besynthesized by a method comprising (a) converting 4-(Boc-amino)pyridineto 4-(Boc-amino)pyridine-3-ethanol, (b) fluorinating4-(Boc-amino)pyridine-3-ethanol by using a [¹⁸F]-containing reagent, and(c) removing Boc to form [¹⁸F]-3-fluoroethyl-4-aminopyridine.

In certain embodiments, the [¹⁸F]-containing reagent includes, but isnot limited to, [¹⁸F]-Kryptofix, [¹⁸F]-F2, [¹⁸F]-AcOF, [¹⁸F]F-TEDA,[¹⁸F]-Benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV) fluoridetrifluoromethanesulfonate, [¹⁸F]-2-fluoroethyl bromide, and[¹⁸F]-fluoromethyl-bromide. In particular aspects, the [¹⁸F] containingreagent is [¹⁸F] Kryptofix.

In some embodiments, an alternative method for producing[¹⁸F]3-fluoro-4-aminopyridine is provided, comprising the steps of (a)using Koser's reagent to iodonate 4-(Boc-amino)pyridin-3-ylboronic acidto form a first intermediate compound, (b) fluorinating the intermediatecompound by using a [¹⁸F]fluor-containing reagent, and (c) using HCl toremove the protecting group to yield [¹⁸F]3-fluoro-4-aminopyridine.

The methods for producing the compounds described herein are not limitedto the exemplary methods described herein. The compounds may besynthesized by any suitable method known in the art and it will beobvious to those skilled in the art that various adaptations, changes,modifications, substitutions, deletions or additions of procedures maybe made without departing from the spirit and scope of the invention.

In certain methods and compositions, embodiments concern the use of acompound for research purposes involving a potassium channel blocker.The compound may be used for its potassium channel blocking activity.Therefore, in some embodiments, methods involve exposing, contacting, oradding a compound discussed herein to a channel or a polypeptideinvolved in channel activity and determining calcium channel activity.In some embodiments, the compound is a control. In other embodiments,the compound is used to screen other compounds for an activity thataffects channel activity (such as by inhibiting or enhancing thatactivity).

Because of the biological activity of the compounds disclosed herein, inadditional embodiments, there are methods and compositions for use ofthese compounds as an avicide. In some embodiments, a compound discussedherein is formulated as grain bait, a powder concentrate or a liquid forexposure to or ingestion by birds. The LD50 for birds is generally inthe range of about 100 parts per million (ppm) to 1000 parts permillion, and dosages are formulated to provide at least that much tobirds. Embodiments also include methods of using an avicide comprisingproviding to an avian an effective amount of a composition comprising acompound discussed herein, including but not limited to those havingFormula I or Formula II. In certain embodiments, providing the compoundcomprises distributing the composition to places that birds can access,including but not limited to distributing it in grass, trees, bushes, onleaves, in bird feeders or in bird baths or other food or water suppliesfor birds. In further embodiments, distributing the composition mayinvolve spraying a liquid or powder composition, or depositing orplacing a solid, liquid or powder composition. In certain embodiments, asubject is a bird.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the measurement orquantitation method.

The use of the word “a” or “an” when used in conjunction with the term“comprising” may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. Compositions and methods“consisting essentially of” any of the ingredients or steps disclosedlimits the scope of the claim to the specified materials or steps whichdo not materially affect the basic and novel characteristic of theclaimed invention.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate the mechanism of action of a potassium channelblocker. (A) shows a scheme of a healthy neuron. (B) shows a scheme of ademyelinated neuron. Aberrant leakage of potassium ions from the axonresults in poor conduction of electrical impulses along the axon. (C)shows a demyelinated neuron treated with a potassium channel blocker.

FIGS. 2A-2G show potassium channel blockers and fluorinated 4-APderivatives. (A) 4-aminopyridine (B) 3,4-diaminopyridine (C)3-methanol-4-aminopyridine (D) 3-fluoro-4-aminopyridine (E)3-fluoromethyl-4-aminopyridine (F) 3-fluoroethyl-4-aminopyridine (G)2-fluoro-4-aminopyridine

FIGS. 3A-3F. (A) shows synthesis of 3-fluoromethyl-4-aminopyridine. (B)shows synthesis of 3-fluoroethyl-4-aminopyridine. (C) shows NMR of3-fluoromethyl-4-aminopyridine. (D) shows high resolution Mass Spectraof 3-fluoromethyl-4-aminopyridine. (E) shows NMR of3-fluoroethyl-4-aminopyridine. (F) shows high resolution Mass spectra of3-fluoroethyl-4-aminopyridine.

FIGS. 4A-4C show enhancement of compound action potential (CAP) by 4-APderivatives. (A) CAP traces before (solid line) and after (dashed line)addition of the drug. Scale bar: 5 mV/5 ms. (B) Relative increase ofmaximum CAP amplitude vs. concentration for each drug. Amplitude wasnormalized to the amplitude before the drug. (C) Half-maximal effectiveconcentration of each molecule and 95% confidence interval obtained fromfitting the data to the Hill equation. n=number of times each drug wastested.

FIGS. 5A-5C show inhibition of ionic current of Shaker K⁺ channel by4-AP derivatives. (A) K⁺ currents were generated by a series of 50 mspulses from −70 mV to +40 mV in increments of 10 mV in the presence ofcumulative concentrations of 4-AP derivatives. Each panel represents theK⁺ current recorded from the same oocyte before and after addition ofthe drug. Scale bar: 1 μA/10 ms. (B) Relative K⁺ current vs.concentration for each drug obtained at +20 mV. (C) Half-maximalinhibitory concentration of each molecule and 95% confidence intervalobtained from fitting the data to the Hill equation. n=number of timeseach drug was tested.

FIGS. 6A-6B show pharmacology of 4-AP derivatives. (A) Pharmacologicalparameters for 4-AP derivatives and control compounds. c Log P:calculated partition coefficient using VCCLAB (I. V. Tetko et al.,Virtual computational chemistry laboratory—design and description.Journal of computer-aided molecular design 19, 453 (June, 2005)), P_(e):permeability coefficient across artificial membrane (n=3), t_(1/2):half-life in mouse microsomes (n=3). (B) Pharmacokinetic profile of 4-APand 3-F-4-AP in plasma and brain of mice after intravenous injection of0.75 mg of drug per mouse kg (n=3 mice per time point).

FIGS. 7A-7E. Brain distribution of 4-AP in mice injected with LPC. Toplabel represents mouse name+section number. (A) Fluorescent microscopyof myelin basic protein (MBP) immunostaining. Each small squarerepresents one picture at 40×. Areas rich in MBP appear darker. Partialdemyelination is evident in certain areas of the corpus callosum. (B)Autoradiography: areas where 4-AP localizes appear darker. 4-AP mostlylocalizes in grey matter areas with almost no signal in white materareas. (C and D) 2× magnification of the corpus callosum area from A andB. Areas of demyelination in the corpus callosum appear darker than therest of the corpus callosum. The corpus callosum has been marked with adashed white line and the areas of demyelination within the corpuscallosum have been circled with a solid white line. Autoradiographicsignal is more intense in areas of demyelination compared to the rest ofthe corpus callosum. Scale bar=2 um. All animals pictured here receivedLPC injections however not all of them showed lesions at the level ofsectioning (ie. no lesions are observed in LPC1-12). (E) Quantificationof the mean pixel intensity in the whole corpus callosum and in thelesion area (as determined by IHC).

FIG. 8. Possible radiosynthesis of [¹⁸F] 3-F-4-AP and [¹⁸F] 3-MeF-4-AP

FIGS. 9A-9B—(A) Experimental scheme: TBI is induced in rats using acontrolled impact. (B) Autoradiography.

DETAILED DESCRIPTION OF THE INVENTION

Multiple sclerosis (MS) is the most common neurodegenerative disease inyoung adults. The progressive demyelination of neurons in the centralnervous system (CNS) is the hallmark of MS (Calabresi, 2007). When axonslose their myelin, K⁺ channels in the axonal membrane become exposed andleak K⁺ ions (FIGS. 1A and B). The aberrant leakage of K⁺ ions from theaxons results in poor impulse conduction, which in turn causes theappearance of neurological symptoms (Ritchie et al., 1981; Waxman andRitchie, 1993; Rasband et al., 1998; Arroyo et al., 2004).

Positron Emission Tomography (PET) allows imaging of molecular changesbefore macroscopic changes have occurred and therefore it provides anopportunity for early detection. It does this by detecting a radiationcoming from a radionuclide introduced in the body in a biologicallyactive molecule that selectively localizes to the area of interest, alsoknown as tracer. Images of the tracer's distribution can bereconstructed using computer analysis allowing precise mapping of itslocation. For example, ¹⁸F-fludeoxyglucose is widely used to imagehighly metabolically active cells such as cancer cells inside anorganism. Similarly, it is conceivable that a PET-active molecule thatselectively localizes to injured areas in the brain could provideaccurate maps of TBI.

During TBI, compression or stretching of the brain often causes axons totear and oligodendrocytes (cells responsible for producing andmaintaining myelin) to break. Injury to oligodendrocytes can leave axonsdevoid of myelin, which then become more sensitive to degeneration. Itis well known that loss of myelin (as in conditions like multiplesclerosis, MS) causes K⁺ channels, which are usually buried beneath themyelin sheath to become exposed and leaky.

4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) are well-knownpotassium channel blockers relatively selective for voltage gated K⁺channels of the K_(v)1 family (Wulff et al., 2009). 4-AP sensitive K+channels, Kv1.1 and Kv1.2, are localized in the juxtaparanodal region ofmyelinated axons. Upon demyelination these channels redistributethroughout the intermodal region of the axons as seen in tissue samplesfrom MS patients and in demyelinated animals. In demyelinated animalsKv1 channels have been shown to be upregulated 2-4 fold. 4-AP and3,4-DAP have been used effectively in the treatment of Lambert-EatonSyndrome and Multiple Sclerosis (Murray and Newsom-Davis, 1981; Soni andKam, 1982; Lundh et al., 1977). 4-AP and 3,4-DAP block K_(v)1 potassiumchannels with affinities in the micromolar range. Binding of 4-AP and3,4-DAP to K_(v)1 potassium channels restores impulse conduction indemyelinated fibers (Yeh et al., 1976; Sherratt et al., 1980; Kirsch andNarahashi, 1978). 4-aminopyridine-3-methanol can also restore impulseconduction of demyelinated fibers (Sun et al., 2010; Leung et al.,2011).

In 2010, the FDA approved a slow-release formulation of 4-aminopyridine(4-AP), to improve walking in MS patients (Ampyra, Acorda Therapeutics,Inc., 2010). 4-AP is a relatively selective blocker of K_(v)1 family ofK⁺ channels (Wulff et al., 2009). The proposed mechanism of action of4-AP in MS patients is that 4-AP blocks K⁺ channels in demyelinatedaxons, which leads to improved impulse conduction.

Fluorinated molecules generally display better pharmacologicalproperties such as increased membrane permeability and metabolicstability than their non-fluorinated analogs. Described herein arecompounds of formula I or II, which contain fluorine and efficientlyblock voltage gated potassium channels. In particular, certainembodiments are directed to fluorinated 4-AP derivatives, such as3-fluoromethyl-4-aminopyridine, or 3-fluoroethyl-4-aminopyridine.

In addition, 4-AP can efficiently cross the blood brain barrier.Application of a computational model for the estimation of log BB (aparameter used to predict blood brain barrier permeability by certaincompounds) predicts that the compounds described herein, in particular,fluorinated 4-AP derivatives, will efficiently cross the blood brainbarrier (Sun, 2004). It has also been shown that 3-F-4-AP is morelipophylic than 4-AP (Arzneimittelforschung, 1989)

4-AP is safe within the concentrations used in therapy, which indicatesthat the compounds described herein, in particular, fluorinated 4-APderivatives are likely to be safe tools when used in humans.

To effectively treat a patient with a neurodegenerative disease, such asMS, it is important to diagnose and evaluate the progression of thedisease in the patient. Currently, magnetic resonance imaging (MRI) isthe primary imaging techniques for the diagnosis and the assessment ofdisorders that disrupt the myelin sheath, including MS. Unfortunately,signal changes on an MRI are non-specific and correlate only indirectlywith the underlying pathology. Moreover, current methods do notcorrelate well with the underlying pathology of the disease and are notwell-suited for use in clinical trials.

PET is a non-invasive medical imaging technique that relies on thedetection of radiation emitted by a radionuclide (radioactive tracer)introduced in the body of the subject on a biologically active molecule.Images of the radioactive tracer's localization can be reconstructed bycomputer analysis providing quantitative maps of the radioactivetracer's distribution in the body of the subject. Such images canprovide valuable information of the biochemistry and physiology of asubject. Because PET is a molecular imaging technique, it can detectcellular abnormalities before anatomical changes have occurred. Forexample, 18F-fluorodeoxyglucose (FDG) is widely used to distinguishhighly metabolically active cancer cells from other cells (Oriuchi etal., 2006). Similarly, it is conceivable that a “PET-active” moleculethat selectively localizes to demyelinated axons could provide accuratemaps of the lesions early in the process.

The most common radioisotopes used in PET are ¹⁸F, ¹⁵O, ¹³N and ¹¹C,with half-lives of 110, 2, 10, and 20 min respectively. ¹⁸F is usuallypreferred due to its longer half-life and its lower positron energywhich results in better resolution. Despite the relatively shorthalf-life of these radioisotopes, they are widely used in medicaldiagnostics as many hospitals have their own cyclotron to prepare theradioactive tracers or have a nearby facility that can prepare theradioactive tracers.

A recent review on PET markers for MS highlighted several potentialtargets for PET imaging including 18 kDa Translocator Protein,Cannabinoid Receptor Type 2, Myelin, Cerebral metabolic rate of glucoseutilization, Type A γ-aminobutyric acid, and Acetyl choline receptor(Owen et al., 2011). Nevertheless, all of these markers havelimitations: some of these tracers were originally developed for otherconditions and suffer from low pathological specificity; others weredeveloped to target myelin or myelin related proteins and have limitedsignal-to-noise ratio and the rest target inflammatory cells which donot necessarily correlate with the underlying demyelination. Morerecently, a report on [¹¹C]PIB, a PET radioactive tracer that binds toamyloid plaques originally developed for Alzheimer's Disease, has beenshown to be useful in quantifying myelin (Stankoff et al., 2011).Nevertheless, since MS is a de-myelinating disorder, it would bedesirable to have access to a PET radioactive tracer specific forde-myelinated axons. In particular, it would be desirable to develop aPET radioactive tracer that targets potassium channels for imagingdemyelination or other conditions.

Incorporation of a positron emitting radionuclide such as ¹⁸F into apotassium channel blocker, such as a 4-AP derivative, allowsvisualization of the location and abundance of exposed potassiumchannels and provides a better assessment of demyelinated regions. Thefact that 4-AP has proven therapeutically beneficial indicates that itpreferentially binds to potassium channels of demyelinated neurons.Furthermore, the fact that there are relatively few side effects of 4-APindicates that there are few off-target receptors at the concentrationscurrently used in therapy. Such properties indicate that the compoundsdescribed herein are suitable for imaging demyelinated neurons withadequate signal-to-noise ratio.

Furthermore, the metabolic stability of [¹⁸F]Fluoroalkylbiphenyls, whichshare a similar core structure to the compounds described herein, havebeen examined and were found to be stable for PET studies (Lee et al.,2004), indicating that the compounds described herein are likely stablefor PET studies.

Substitution of ¹⁸F for OH or H is common in the art. Such substitutionsgenerally preserve the biological properties of the molecule and renderthe molecules suitable for imaging using PET or SPECT cameras. Forexample substitution of the OH in position 2 of glucose with ¹⁸F doesnot alter the capability to be uptaken by cells. Many examples of ¹⁸Fsubstitutions that preserve the parent molecule's properties can befound on the MICAD database (available on the world wide web atncbi.nlm.nih.gov/books/NBK5330/).

FIG. 1C shows a cartoon representation of the proposed mechanism ofaction of the radioactive tracer. 4-AP as well as the radioactivetracers described herein bind to potassium channels on demyelinatedaxons decreasing efflux of K⁺. Visualization of the localization ofthese molecules can inform of the localization and extent ofdemyelinated axons.

Disclosed herein are new radioactive tracers for PET, which serve asnovel diagnostic markers to image demyelinated axons in a subject. Inparticular embodiments, the new radioactive tracers for PET are¹⁸F-labeled versions of 4-AP derivatives. Methods for their manufactureand methods for their use in in vivo imaging of the central nervoussystem to diagnose and/or assess the progression of MS or other diseasesare also provided. The present disclosure also provides fluorinecontaining compounds that bind to potassium channels, methods for theirmanufacture and methods for their use in the treatment ofneurodegenerative diseases.

I. DEFINITIONS

The term “radioactive isotope” refers to an isotope having an unstablenucleus that decomposes spontaneously by emission of a nuclear electron,positron, or helium nucleus and radiation, thus achieving a more stablenuclear composition.

The term “deuterated version” as used herein means one or more ofhydrogen in a compound is replaced with ²H, an isotope of hydrogen.

As used herein, the term “radioactive tracer”, or “radioactive label”,or “tracer”, or “radiotracer” means a chemical compound in which one ormore atoms have been replaced by a radioisotope. By virtue of itsradioactivity, it can be used to explore the mechanism of chemicalreactions by tracing the path that the radioisotope follows fromreactants to products. A radioactive tracer can also be used to trackthe distribution of a substance within a natural system such as a cellor tissue. Radioactive tracers form the basis of a variety of imagingsystems, such as PET scans and SPECT scans.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present invention. The prodrug itselfmay or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2n, where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diasteromers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤15%, morepreferably ≤10%, even more preferably ≤5%, or most preferably ≤1% ofanother stereoisomer(s).

“Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

II. COMPOUNDS THAT BLOCK POTASSIUM CHANNELS

Certain embodiments provide compounds that block potassium channelshaving the following formula:

wherein R₁, R₂, R₃, and R₄ are independently selected from the groupconsisting of H, (CH₂)_(n)X, CH₂OCH₂CH₂X, NH₂, CH₂OH, and CF₃; and R₅ isselected from the group consisting of H, (CH₂)_(m)X, and OH, whereinn=0, 1, 2, 3, 4, or 5, and m=2, 3, 4, or 5; wherein X represents afluorine atom or an isotope thereof, wherein at least one of R₁, R₂, R₃,R₄ and R₅ is not hydrogen; wherein at least one of R₁, R₂, R₃, R₄ and R₅contains a fluorine atom or an isotope thereof; wherein when R₂ is NH₂or CH₂OH or a nonradioactive fluorine or CF₃, at least one of R₁, R₃,R₄, and R₅ is not hydrogen; wherein when R₄ is NH₂, CH₂OH, anonradioactive fluorine, or CF₃, at least one of R₁, R₂, R₄, and R₅ isnot hydrogen; and wherein any of C, N, O is optionally replaced by theisotope ¹¹C, ¹³N, ¹⁵O, respectively, or a pharmaceutical acceptable saltthereof, a tautomer thereof or a deuterated version thereof.

In some embodiments, the compounds have the formulas found in FIGS.2A-G. In particular embodiments, the compounds have the formulas of FIG.2E and FIG. 2F, which are not commercially available and have never beendescribed before.

In further embodiments, 4-AP derivatives having the following formulaare provided:

wherein M is (CH₂)_(n)Y, and wherein n=0, 1, or 2, and Y is fluorine oran isotope thereof.

In certain embodiments, there are provided compounds of formula (III):

wherein R is selected from the group consisting of CH₃, CH₂F, CHF₂, andCF₃, and wherein C is substituted by ¹¹C or at least one of F issubstituted by ¹⁸F in R.

Further embodiments are directed to the compounds of formula (IV):

wherein R is selected from the group consisting of CF₃, CH₂F, CH₃CH₂F,C(CH₃)₃, and wherein at least one of F or H in the R group issubstituted by ¹⁸F.

The compounds provided by the present disclosure are described in thesummary of the invention section and in the claims below.

Compounds employed in methods described herein may contain one or moreasymmetrically-substituted carbon or nitrogen atoms, and may be isolatedin optically active or racemic form. Thus, all chiral, diastereomeric,racemic form, epimeric form, and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. Compounds may occur as racemates andracemic mixtures, single enantiomers, diastereomeric mixtures andindividual diastereomers. In some embodiments, a single diastereomer isobtained. The compounds can be formulated as a mixture of one or morediastereomers. Alternatively, the diastereomers can be separated and oneor more of the diastereomers can be formulated individually. The chiralcenters of the compounds disclosed herein can have the S or the Rconfiguration, as defined by the IUPAC 1974 Recommendations. Forexample, mixtures of stereoisomers may be separated using techniquesknown to those of skill in the art.

Atoms making up the compounds of the present invention are intended toinclude all isotopic forms of such atoms. Compounds of the presentinvention include those with one or more atoms that have beenisotopically modified or enriched, in particular those withpharmaceutically acceptable isotopes or those useful for pharmaceuticalresearch. Isotopes, as used herein, include those atoms having the sameatomic number but different mass numbers. By way of general example andwithout limitation, isotopes of hydrogen include deuterium and tritium,and isotopes of carbon include ¹¹C, ¹³C and ¹⁴C. Similarly, it iscontemplated that one or more carbon atom(s) of a compound of thepresent invention may be replaced by a silicon atom(s). Furthermore, itis contemplated that one or more oxygen atom(s) of a compound of thepresent invention may be replaced by a sulfur or selenium atom(s).

Compounds disclosed herein may also exist in prodrug form. Sinceprodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing,etc.), the compounds employed in some methods of the invention may, ifdesired, be delivered in prodrug form. Thus, certain embodimentscontemplate prodrugs of compounds described herein as well as methods ofdelivering prodrugs. Prodrugs of the compounds may be prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound. Accordingly, prodrugs include, forexample, compounds described herein in which a hydroxy, amino, orcarboxy group is bonded to any group that, when the prodrug isadministered to a subject, cleaves to form a hydroxy, amino, orcarboxylic acid, respectively.

It should be recognized that the particular anion or cation forming apart of any salt of this invention is not critical, so long as the salt,as a whole, is pharmacologically acceptable. Additional examples ofpharmaceutically acceptable salts and their methods of preparation anduse are presented in Handbook of Pharmaceutical Salts: Properties, andUse (2002), which is incorporated herein by reference.

It should be further recognized that the compounds of the presentinvention include those that have been further modified to comprisesubstituents that are convertible to hydrogen in vivo. This includesthose groups that may be convertible to a hydrogen atom by enzymologicalor chemical means including, but not limited to, hydrolysis andhydrogenolysis. Examples include hydrolyzable groups, such as acylgroups, groups having an oxycarbonyl group, amino acid residues, peptideresidues, o-nitrophenylsulfenyl, trimethylsilyl, tetrahydropyranyl,diphenylphosphinyl, and the like. Examples of acyl groups includeformyl, acetyl, trifluoroacetyl, and the like. Examples of groups havingan oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl(—C(O)OC(CH₃)₃), benzyloxycarbonyl, p-methoxy-benzyloxycarbonyl,vinyloxycarbonyl, β-(p-toluenesulfonyl)ethoxycarbonyl, and the like.Suitable amino acid residues include, but are not limited to, residuesof Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp(aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine),Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe(phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp(tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse(homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn(ornithine) and β-Ala. Examples of suitable amino acid residues alsoinclude amino acid residues that are protected with a protecting group.Examples of suitable protecting groups include those typically employedin peptide synthesis, including acyl groups (such as formyl and acetyl),arylmethoxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Suitable peptide residues include peptide residuescomprising two to five amino acid residues. The residues of these aminoacids or peptides can be present in stereochemical configurations of theD-form, the L-form or mixtures thereof. In addition, the amino acid orpeptide residue may have an asymmetric carbon atom. Examples of suitableamino acid residues having an asymmetric carbon atom include residues ofAla, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr. Peptideresidues having an asymmetric carbon atom include peptide residueshaving one or more constituent amino acid residues having an asymmetriccarbon atom. Examples of suitable amino acid protecting groups includethose typically employed in peptide synthesis, including acyl groups(such as formyl and acetyl), arylmethoxycarbonyl groups (such asbenzyloxycarbonyl and p-nitrobenzyloxycarbonyl), tert-butoxycarbonylgroups (—C(O)OC(CH₃)₃), and the like. Other examples of substituents“convertible to hydrogen in vivo” include reductively eliminablehydrogenolyzable groups. Examples of suitable reductively eliminablehydrogenolyzable groups include, but are not limited to, arylsulfonylgroups (such as o-toluenesulfonyl); methyl groups substituted withphenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl);arylmethoxycarbonyl groups (such as benzyloxycarbonyl ando-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl groups (such asβ,β,β-trichloroethoxycarbonyl and β-iodoethoxycarbonyl).

The compounds described herein may exist in unsolvated forms as well assolvated forms, including hydrated forms. In general, the solvated formsare equivalent to unsolvated forms and are within the scope of thecompounds described herein. The compounds described herein may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses described herein and are intended to bewithin the scope of the compounds described herein.

Compounds provided herein may also have the advantage that they may bemore efficacious than, be less toxic than, be longer acting than, bemore potent than, produce fewer side effects than, be more easilyabsorbed than, and/or have a better pharmacokinetic profile (e.g.,higher oral bioavailability and/or lower clearance) than, and/or haveother useful pharmacological, physical, or chemical properties over,compounds known in the prior art, whether for use in the indicationsstated herein or otherwise.

III. FORMULATIONS

The compounds described herein can be formulated for enteral,parenteral, topical, or pulmonary administration. In other embodiments,the formulation is for administration to a subject, but it may not bedirectly to the subject. The compounds can be combined with one or morepharmaceutically acceptable carriers and/or excipients that areconsidered safe and effective and may be administered to an individualwithout causing undesirable biological side effects or unwantedinteractions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients.

A. Parenteral Formulations

The compounds described herein can be formulated for parenteraladministration. “Parenteral administration”, as used herein, meansadministration by any method other than through the digestive tract ornon-invasive topical or regional routes. For example, parenteraladministration may include administration to a patient intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostatically, intrapleurally,intratracheally, intravitreally, intratumorally, intramuscularly,subcutaneously, subconjunctivally, intravesicularly, intrapericardially,intraumbilically, by injection, and by infusion.

Parenteral formulations can be prepared as aqueous compositions usingtechniques is known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the active compounds as the free acid orbase or pharmacologically acceptable salts thereof can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents, andcombination thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium oflong chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

Formulations may be stable over a period of 6 months when stored at roomtemperature or 4° C.

B. Enteral Formulations

Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art.

Formulations may be prepared using a pharmaceutically acceptablecarrier. As generally used herein “carrier” includes, but is not limitedto, diluents, preservatives, binders, lubricants, disintegrators,swelling agents, fillers, stabilizers, and combinations thereof.

Carrier also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release dosage formulations may be prepared asdescribed in standard references such as “Pharmaceutical dosage formtablets” (1989), “Remington—The science and practice of pharmacy”(2000), and “Pharmaceutical dosage forms and drug delivery systems”(1995). These references provide information on carriers, materials,equipment and process for preparing tablets and capsules and delayedrelease dosage forms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients include, but are notlimited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions. Suitablestabilizers include, but are not limited to, antioxidants, butylatedhydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E,tocopherol and its salts; sulfites such as sodium metabisulphite;cysteine and its derivatives; citric acid; propyl gallate, and butylatedhydroxyanisole (BHA).

Oral dosage forms, such as capsules, tablets, solutions, andsuspensions, can for formulated for controlled release. For example, theone or more 4-AP derivatives and optional one or more additional activeagents can be formulated into nanoparticles, microparticles, andcombinations thereof, and encapsulated in a soft or hard gelatin ornon-gelatin capsule or dispersed in a dispersing medium to form an oralsuspension or syrup. The particles can be formed of the drug and acontrolled release polymer or matrix. Alternatively, the drug particlescan be coated with one or more controlled release coatings prior toincorporation in to the finished dosage form.

In another embodiment, the one or more 4-AP derivatives and optional oneor more additional active agents are dispersed in a matrix material,which gels or emulsifies upon contact with an aqueous medium, such asphysiological fluids. In the case of gels, the matrix swells entrappingthe active agents, which are released slowly over time by diffusionand/or degradation of the matrix material. Such matrices can beformulated as tablets or as fill materials for hard and soft capsules.

In still another embodiment, the one or more 4-AP derivatives, andoptional one or more additional active agents are formulated into a soldoral dosage form, such as a tablet or capsule, and the solid dosage formis coated with one or more controlled release coatings, such as adelayed release coatings or extended release coatings. The coating orcoatings may also contain the 4-AP derivatives and/or additional activeagents.

C. Topical Formulations

Suitable dosage forms for topical administration include creams,ointments, salves, sprays, gels, lotions, emulsions, and transdermalpatches. The formulation may be formulated for transmucosal,transepithelial, transendothelial, or transdermal administration. Thecompounds can also be formulated for intranasal delivery, pulmonarydelivery, or inhalation. The compositions may further contain one ormore chemical penetration enhancers, membrane permeability agents,membrane transport agents, emollients, surfactants, stabilizers, andcombination thereof.

D. Other Formulations

Any of the formulations discussed above may be used for a formulationthat is not a pharmaceutical formulation. In some embodiments, aformulation may be prepared for administration to a subject by a methodthat is direct or by a method that is indirect. In certain embodiments,a compound is provided in a liquid, solid, or powder formulation. Thecompound may be in a composition that is sprayed or otherwise applied toa surface or location. In some embodiments, the composition is placed ina location that is accessible to the subject so that the subject ingestsor comes into contact with the composition. In certain embodiments, thecompound is absorbed or adsorbed by the subject.

IV. METHODS OF MAKING COMPOUNDS THAT BLOCK POTASSIUM CHANNELS

The compounds provided by the present disclosure are described in thesummary of the invention section and in the claims below. They may bemade using the methods outlined in the summary of the invention sectionand in the Examples section. These methods can be further modified andoptimized using the principles and techniques of organic chemistry asapplied by a person skilled in the art. Such principles and techniquesare taught, for example, in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure (2007), which is incorporated byreference herein.

The compounds or compositions described herein can also be prepared byany of the applicable techniques of organic synthesis and polymerchemistry. Many such techniques are well known in the art. Many of theknown techniques are elaborated in Compendium of Organic SyntheticMethods, Vol 1, 1971; Vol. 2, 1974; Vol. 3, 1977; Vol. 4, 1980; Vol. 5,1984; and Vol. 6, 1985; Comprehensive Organic Synthesis Selectivity,Strategy & Efficiency in Modern Organic Chemistry, 1993; AdvancedOrganic Chemistry, Part B: Reactions and Synthesis, 4^(th) Ed., 2001;Advanced Organic Chemistry, Reactions, Mechanisms, and Structure, 2^(nd)Ed., 1977; Protecting Groups in Organic Synthesis, 2^(nd) Ed., 991;Comprehensive Organic Transformations, 2^(nd) Ed., 1999, Textbook ofPolymer Chemistry, 3^(rd) Ed., 1984, Organic Polymer Chemistry,2^(nd)Ed., 1973, and Polymer Science, 1986. These are incorporatedherein by reference.

V. METHODS OF USING COMPOUNDS THAT BLOCK POTASSIUM CHANNELS

A. Treatment

The compounds described herein can be administered to provide aneffective amount to prevent, treat or mitigate a symptom of a variety ofdiseases and disorders, in particular, a disease associated withdemyelination, such as multiple sclerosis. The compounds describedherein can be administered to a subject in need thereof to treat thesubject either prophylactically (e.g., to prevent a demyelinationdisease) or therapeutically (e.g., to treat a demyelination diseaseafter it has been detected), including, but not limited to, amelioratingthe symptoms of a disease, reducing the pain of the patient, delayingthe progression of the disease, preventing new attacks or recurring ofthe disease, preventing disability and/or increasing survival time ofthe patient.

The compounds described herein can bind to potassium channels, such asKv1 potassium channels located in the axonal membrane to partially orcompletely restore the impulse conduction along the axon. In someembodiments, the compounds described herein may also be used to treatnon-neurological diseases when blocking of potassium channels in theheart or other tissues expressing potassium channels is desired.

By administering the compounds described herein to a patient sufferingfrom a demyelinating disease, one or more symptoms associated withdemyelination may be mitigated or eliminated. The symptoms includechanges in sensation such as loss of sensitivity or tingling, prickingor numbness (hypoesthesia and paresthesia), muscle weakness, clonus,muscle spasms, or difficulty in moving; difficulties with coordinationand balance (ataxia); problems in speech (dysarthria) or swallowing(dysphagia), visual problems (nystagmus, optic neuritis includingphosphenes, or diplopia), fatigue, acute or chronic pain, and bladderand bowel difficulties. The symptoms may further include cognitiveimpairment of varying degrees and emotional symptoms of depression orunstable mood are also common, Uhthoffs phenomenon, an exacerbation ofextant symptoms due to an exposure to higher than usual ambienttemperatures, and Lhermitte's sign, an electrical sensation that runsdown the back when bending the neck.

Exemplary demyelination diseases which can be treated by the compoundsdescribed herein include, but are not limited to, multiple sclerosis,spinal cord compression, ischemia, acute disseminated encephalomyelitis,optic neuromyelitis, leukodystrophy, progressive multifocalleukoencephalopathy, metabolic disorders, toxic exposure, congenitaldemylinating disease, peripheral neuropathy, encephalomyelitis, centralpontine myelolysis, Anti-MAG Disease, Guillain-Barre syndrome, chronicinflammatory demyelinating polyneuropathy, or multifocal motorneuropathy (MMN). Exemplary leukodystrophy includes, but is not limitedto adrenoleukodystrophy, Alexander's Disease, Canavan Disease, KrabbeDisease, Metachromatic Leukodystrophy, Pelizaeus-Merzbacher Disease,vanishing white matter disease, Refsum Disease, Cockayne Syndrome, Vander Knapp Syndrome, or Zellweger Syndrome.

Patients can be treated using a variety of routes of administrationincluding systemic administration, such as intravenous administration orsubcutaneous administration, oral administration or by intratumoralinjection.

In certain embodiments, it may be desirable to provide continuousdelivery of one or more compounds described herein to a patient in needthereof. For intravenous or intraarterial routes, this can beaccomplished using drip systems, such as by intravenous administration.For topical applications, repeated application can be done or a patchcan be used to provide continuous administration of the compoundsdescribed herein, including 4-AP derivatives over an extended period oftime. Extended release formulations can also be used to provide limitedbut stable amounts of the drug over an extended period of time.

For internal applications, continuous perfusion of the region ofinterest may be desirable. This could be accomplished bycatheterization, post-operatively in some cases, followed by continuousadministration of the one or more 4-AP derivatives. The time period forperfusion can be readily determined by the attending physician clinicianfor a particular subject. Perfusion times typically range from about 1-2hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about1-2 days, to about 1-2 weeks or longer. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by single or multiple injections, adjusted for the period oftime over which the injections are administered.

The compositions described herein contain an effective amount of the oneor more compounds described herein. The amount to be administered can bereadily determined by the attending physician based on a variety offactors including, but not limited to, age of the patient, weight of thepatient, disease or disorder to be treated, presence of a pre-existingcondition, and dosage form to be administered (e.g., immediate releaseversus modified release dosage form). Typically, the effective amount isfrom about 0.01 mg/kg/day to about 100 mg/kg/day, from 0.1 mg/kg/day to50 mg/kg/day, from 0.1 mg/kg/day to 25 mg/kg/day, 0.1 mg/kg/day to 10mg/kg/day, from 0.1 mg/kg/day to 1 mg/kg/day or any range derivabletherein. Dosages greater or less than this may be administered dependingon the diseases or disorder to be treated.

The therapeutically effective doses could also be determined by using ananimal model. For example, a mouse bearing experimental autoimmuneencephalomyelitis (EAE) could be used to optimize appropriatetherapeutic doses prior to translating to a clinical environment.

The therapeutically effective doses could also be determined by using ananimal model. For example, a rodent bearing Traumatic Brain Injury couldbe used to optimize appropriate therapeutic doses prior to translatingto a clinical environment.

In some embodiments, the compounds and compositions disclosed herein maybe useful in a variety of manners. In some embodiments, the compoundsand compositions disclosed herein may be useful for improving gait instroke patients. In some embodiments, the compounds and compositionsdisclosed herein may be useful in research to induce seizures. In someembodiments, the compounds and compositions disclosed herein may beuseful as pest control agents. In some embodiments, the compounds andcompositions disclosed herein may be useful for Parkinson's Disease,pediatric and adult Cerebral Palsy, Spinal Cord Injury, Lambert Eatonsyndrome, and MS.

In some embodiments, derivatives of 4-AP will have improvedpharmacological properties over 4-AP. 3-F-4-AP has better permeabilityinto the CNS than 4-AP and, therefore, it may be better for CNSdiseases. Fluorinated 4-APs may have longer half life than 4-AP, may beless toxic, and may be more stable to metabolic degradation.

B. Imaging

The compounds described herein can also be used as imaging agents inmedical imaging applications. Medical imaging is the technique andprocess used to create images of the human body (or parts and functionthereof) for clinical purposes (medical procedures seeking to reveal,diagnose or examine disease) or medical science (including the study ofnormal anatomy and physiology). Medical imaging may also be applied toan animal body. Commonly used medical imaging techniques include, butare not limited to, radiography, magnetic resonance imaging (MRI),fiduciary markers, nuclear medicine, photo acoustic imaging, breastthermography, tomography, and ultrasound.

1. Radiography

Projection radiograph, also known as x-rays, and fluoroscopy are twoforms of radiographic images used in medical imaging; with the latterbeing useful for catheter guidance. This imaging modality utilizes awide beam of x rays for image acquisition and is the first imagingtechnique available in modern medicine.

2. Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging instrument (MRI scanner), or “nuclearmagnetic resonance (NMR) imaging” scanner as it was originally known,uses powerful magnets to polarise and excite hydrogen nuclei (singleproton) in water molecules in human tissue, producing a detectablesignal which is spatially encoded, resulting in images of the body.

3. Fiduciary Markers

Fiduciary markers are used in a wide range of medical imagingapplications. Images of the same subject produced with two differentimaging systems may be correlated (called image registration) by placinga fiduciary marker in the area imaged by both systems. In this case, amarker which is visible in the images produced by both imagingmodalities must be used.

4. Nuclear Medicine

Nuclear medicine encompasses both diagnostic imaging and treatment ofdisease. Nuclear medicine uses certain properties of isotopes and theenergetic particles emitted from radioactive material to diagnose ortreat various pathology. This approach is often used in e.g.,scintigraphy, SPECT and PET to detect regions of biologic activity thatmay be associated with disease. Isotopes are often preferentiallyabsorbed by biologically active tissue in the body, and can be used toidentify tumors or fracture points in bone. Images are acquired aftercollimated photons are detected by a crystal that gives off a lightsignal, which is in turn amplified and converted into count data.

Scintigraphy (“scint”) is a form of diagnostic test whereinradioisotopes are taken internally, for example intravenously or orally.Then, gamma cameras capture and form two-dimensional images from theradiation emitted by the radiopharmaceuticals.

SPECT is a 3D tomographic technique that uses gamma camera data frommany projections and can be reconstructed in different planes. In SPECTimaging, the patient is injected with a radioisotope, most commonlyThallium 201TI, Technetium 99mTC, Iodine 123I, and Gallium 67Ga.

Positron emission tomography (PET) uses coincidence detection to imagefunctional processes. Short-lived positron emitting isotopes, such as¹⁸F, are incorporated with an organic substance such as glucose,creating F18-fluorodeoxyglucose, which can be used as a marker ofmetabolic utilization. Images of activity distribution throughout thebody can show rapidly growing tissue, like tumor, metastasis, orinfection. PET images can be viewed in comparison to computed tomographyscans to determine an anatomic correlate. Modern scanners combine PETwith a CT, or even MRI, to optimize the image reconstruction involvedwith positron imaging. This is performed on the same equipment withoutphysically moving the patient off of the gantry. The resultant hybrid offunctional and anatomic imaging information is a useful tool innon-invasive diagnosis and patient management.

5. Tomography

Tomography is the method of imaging a single plane, or slice, of anobject resulting in a tomogram. There are several forms of tomography,including linear tomography, poly tomography, zonography,orthopantomograph (OPT or OPG), and computed tomography (CT).

6. Ultrasound

Medical ultrasonography uses high frequency broadband sound waves in themegahertz range that are reflected by tissue to varying degrees toproduce (up to 3D) images. This is commonly associated with imaging thefetus in pregnant women. Uses of ultrasound are much broader, however.Other important uses include imaging the abdominal organs, heart,breast, muscles, tendons, arteries and veins.

In certain embodiments, the compounds described herein are used for invivo imaging of the central nervous system. More specifically, thecompounds described herein bind to potassium channels, including Kv1channels. The compounds disclosed herein contain one or moreradioisotopes. Exemplary radioisotopes include, but are not limited to,¹⁸F, ¹¹C, ¹³N, and ¹⁵O. It would be within an artisan's ordinary skillto choose appropriate radioisotope suitable for the imaging techniqueintended to use. In some embodiments, one or more imaging techniques maybe combined for imaging purposes. For example, PET may be combined withMRI. In some aspects, PET is used to image demyelination and MRI is usedto image inflammation. PET and MRI are complement to each other and canprovide valuable information on progression of the MS disease in apatient.

One particular embodiment is directed to radiolabelled 4-AP derivativesthat target potassium channels of demyelinated neurons. Theseradiotracers may be used as in vivo imaging agents for demyelination. Inparticular embodiments, these radiotracers are suitable for PET imagingtechnique. In one embodiment, the radiotracers described herein contain¹⁸F.

Since the compounds described herein are capable of blocking potassiumchannels, such as Kv1 potassium channels located in the axonal membrane,the use of ¹⁸F-labeled 4-AP derivatives such as[¹⁸F]-3-fluoromethyl-4-aminopyridine, or [¹⁸F]-3-fluoro-4-aminopyridine,or other radiotracers described herein allows visualization ofdemyelinated axons in live animals by proper medical imaging techniques,such as PET. Therefore, the compounds described herein may be used todiagnose a demyelinating disease or assessing the progression of ademyelinating disease by administering the compounds to a subject inneed thereof and detecting the compounds in the subject by propermedical imaging technique, such as PET, PET-Time-Activity Curve (TAC),PET-MRI, in particular, PET. In some embodiments, one or more medicalimaging techniques disclosed herein may be used to diagnose or evaluatethe progression of a disease.

VI. KITS

In various aspects, a kit is envisioned containing one or more compoundsdescribed herein. The kit may contain one or more sealed containers,such as a vial, containing any of the compounds described herein and/orreagents for preparing any of the compounds described herein. In someembodiments, the kit may also contain a suitable container means, whichis a container that will not react with components of the kit, such asan Eppendorf tube, an assay plate, a syringe, a bottle, or a tube. Thecontainer may be made from sterilizable materials such as plastic orglass.

The kit may further include instructions that outline the proceduralsteps for methods of treatment or prevention of disease, and will followsubstantially the same procedures as described herein or are known tothose of ordinary skill. The instruction information may be in acomputer readable media containing machine-readable instructions that,when executed using a computer, cause the display of a real or virtualprocedure of delivering a pharmaceutically effective amount of one ormore compounds described herein.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the claims.

Example 1. Synthesis of Fluorinated Derivative of 4-AP

4-AP (FIG. 2A) is a relatively specific blocker of voltage-gated K⁺channels (K_(v)1 family). 4-AP is a membrane permeable molecule thatbinds to the intracellular mouth of the K⁺ channel blocking ioniccurrents (Sherratt et al., 1980). In demyelinated axons these channelsare exposed and easily accessible to the drug. For this reason, theinventors believe that labeling 4-AP with a positron emittingradionuclide will enable imaging of demyelinated regions.

Furthermore, since Kv1 channels are upregulated 2-4 fold in demyelinatedanimals such as shiverer mice and upregulation of Kv1 channels indemyelinated axons suggests greater signal, the inventors expect thatthe signal proceeding from demyelinated axons will be greater.

A common approach in the design of PET radioactive tracers is to replacea hydrogen (H), hydroxyl (OH) or methyl (CH₃) group with fluorine-18(Ametamey et al., 2008). Fluorine-18 is the preferred radionuclide dueto its low positron energy and its longer half-life. The low positronenergy gives it greater spatial resolution and its longer half-lifefacilitates off-site production and distribution.

In an effort to design 4-AP derivatives that maintain activity and aresuitable for PET imaging, the inventors began by examining 4-APderivatives that are known to block K⁺ channels. 4-AP (FIG. 2A) and3,4-diaminopyridine (FIG. 2B), are well-known K⁺ channel blockersdiscovered in the 1970's (Sherratt et al., 1980; Kirsch and Narahashi,1978). More recently Shi et al described 3-methanol-4-aminopyridine(FIG. 2C) as a novel K⁺ channel blocker (Sun et al., 2010; Leung et al.,2011). From these structures, it appears that some variation ispermitted on the 3 position of the pyridine ring. It has also beensuggested that certain variations on the 4 position of the pyridine 4appear to be acceptable as well (Smith et al., 2005). Thus, theinventors hypothesized that 3-fluoro-4-aminopyridine (FIG. 2D),3-fluoromethyl-4-aminopyridine (FIG. 2E) and3-fluoroethyl-4-aminopyridine (FIG. 2F) could be suitable K⁺ channelblockers. These structures (FIGS. 2D-2F) contain different substitutionson position 3 of the pyridine ring which does not alter its function.The inventors also proposed that compound 2-fluoro-4-aminopyridine (FIG.2G) could also bind to K⁺ channels.

Before radioactive labeling, it is important to test whether thesefluorinated derivatives are still able to bind to K⁺ channels.Non-radioactive 3-fluoro-4-aminopyridine (FIG. 2D) is commerciallyavailable from Sigma. Synthesis of fluorinated pyridines which share acore structure similar to the compounds described herein has beenreported previously (Lee and Chi, 1999; Mobinikhaledi and Foroughifar,2006). However, 3-fluoromethyl-4-aminopyridine (FIG. 2E) and3-fluoroethyl-4-aminopyridine (FIG. 2F) have never been made before.These compounds were synthesized according to syntheses outlined inFIGS. 3A-3B. The production of final products3-fluoromethyl-4-aminopyridine and 3-fluoroethyl-4-aminopyridine areverified by NMR (FIGS. 3C, 3E) and high resolution Mass Spectra (FIGS.3D, 3F), respectively.

Example 2. Confirm Binding to Potassium Channels

In order to confirm that the fluorinated 4-AP derivatives maintain theability to block K⁺ channels and select the most suitable compound forimaging studies, the inventors measured inhibition of K⁺ currents inShaker channels (Kv1.2) using cut-open oocyte voltage clamp (Stefani andBezanilla, 1998) (FIGS. 5A-5B). Using the cut-open oocyte voltage clampstudies to screen compounds to determine their potency towards Kv1cannels was previously described in Starace and Bezanilla et al. (2004).

Remarkably, 3-fluoro-4-aminopyridine (3F-4AP) has very similar affinityto 4-AP for K⁺ channels. 3-fluoromethyl-4-aminopyridine (3MeF-4AP) issimilar to 3-methanol-4-aminopyridine (3MeOH-4AP) and around 10-foldless potent than 4-AP. 3-fluoroethyl-4-aminopyridine (3EtF-4AP) and2-fluoro-4-aminopyridine (2F-4AP) are at least a hundred fold-lesspotent than 4-AP. Based on these results, 3F-4AP and 3-MeF-4AP are thepreferred molecules for imaging and therapy.

One advantage of 4-AP, and presumably of its analogs, is that they bindto all channels from the K_(v)1 family. It is known that neurons expressseveral of these channels (Kv1.1, K_(v)1.2, K_(v)1.4, K_(v)1.5,K_(v)1.6, K_(v)1.7 and K_(v)1.8, among which the most important neuronalvoltage gated K+ channels are Kv1.1 and Kv1.2) and that these channelscan form hererotetramers. However, it is unclear which one or severalare responsible for the aberrant efflux of K⁺ ions from demyelinatedaxons and thus, a broad-spectrum channel may be beneficial.

A desired property for radioactive tracers is high affinity. It isstriking that 4-AP and 3,4-diaminopyridine possess a relatively modestaffinity to K⁺ channels (μM to mM) and yet that they are useful intherapeutics (Murray and Newsom-Davis, 1981; Maddison and Newsom-Davis,2003; Goodman et al., 2009). It is possible that these molecules have ahigher effective affinity in vivo as they bind quasi-irreversibly to thechannel. Once bound to the channel these molecules become trapped insidethe channel and do not dissociate. Thus, it is expected that despitetheir modest affinity, the PET markers described herein will display ahigh signal-to-noise ratio.

It is important to note that even though the in vitro affinity of 4-APis low (˜250 μM), 4-AP is active at much lower concentrations in vivo(˜0.5 μM). This difference might be because blockage of a small fractionof channels already leads to an effect and because 4-AP binds to thechannels when they are open and once the channels close, 4-AP becomestrapped inside, functioning as a non-reversible ligand (Armstrong andLoboda, 2001).

Example 3. In Vivo Effects of Fluorinated 4-AP Derivative

A consequence of excessive K⁺ channel blockage is the advent ofseizures. 4-AP is known to cause seizures in mice at high doses. Theinventors tested some 4-AP derivatives described herein and comparedthem to 4-AP in their ability to cause seizures (Table 1).

The fact that only the molecules that are active by cut-open voltageclamp are able to cause seizures strongly suggests that these moleculesare targeting K+ channels receptors in vivo.

In addition, the inventors noticed that Shiverer mice, which harbor amutation on myelin basic protein and suffer from demyelination of theCNS, appear to be less sensitive to 4-AP induced seizures. Previousstudies have shown that Shiverers and other demyelinated mice display anabnormal localization pattern of K_(v)1 channels and a 2-4 fold increasein expression of K_(v)1.1 and K_(v)1.2 channels in axons (Wang et al.,1995). The inventors believe that the higher expression of K_(v)1channels in Shiverer mice is the reason for why these animals are lesssensitive to 4-AP. It is not known whether K_(v)1 channels are alsoupregulated in MS patients, but it is known that K_(v)1 channels in MSlesions present a similar localization patterns as in demyelinatedanimals (Coman et al., 2006). Therefore, similar upregulation isanticipated. Accordingly, upregulation of K_(v)1 channels in MS patientlesions combined with the presumed lower accessibility of 4-AP to K_(v)1channels in myelinated axons make K_(v)1 channels an attractive targetfor PET imaging.

At high doses, 4-AP causes tremors, muscle spasms and seizures. Asummary of the observations is shown in Table 1.

TABLE 1 Effects after intraperitoneal injection of 4-AP derivatives (100μL per 10 g of mouse) Dose MW μmol/ mg/ Drug g/mol kg kg Effect 4-AP94.11 30 2.82 Mild tremor, mouse quiet 60 5.65 Severe tremor, mildjerks, salivation 90 8.47 Severe tremors and seizures that start 10 minpost-injection and last ca. 2 h 3-F-4-AP 112.11 30 3.36 Mouse quiet 606.73 Mild tremor, mouse quiet 90 10.1 Severe tremors and seizures thatstart 10 s post-injection and last ca. 30 min. 1 of 5 mice died ofseizure 3-MeF-4-AP 126.13 30 3.78 No effect 60 7.57 Very mild tremor.Normal after 30 min 90 11.4 Tremor and occasional jerks that last ca. 45min 120 15.1 Severe tremor and seizures that start 5 min post-injectionand last ca. 45 min. 1 of 5 mice died of seizure 3-MeOH-4-AP 124.14 960119.1 No effect 2-F-4-AP 112.11 1920 215.2 No effect N = 5 per group.

From this experiment it can be seen that 3-F-4-AP and 3-MeF-4-AP havevery similar effects as 4-AP in mice. Both of these drugs causesalivation, tremors, jerks, extension of the hind limbs and seizures.3-F-4-AP has very similar potency to that of 4-AP and acts much faster(onset of seizures at highest dose 10 s vs. 10 min) which is consistentwith a faster absorption and a higher permeation of the blood-brainbarrier. 3-MeF-4-AP is slightly less potent than 4-AP but remarkablypotent considering that in the voltage-clamp and optic nerve experimentsit was found to be 6-20 times less potent than 4-AP. In contrast,2-F-4-AP and 3-MeOH-4-AP did not cause any effects at doses up to 20times higher. The inventors also tested the effect of the drugs given byoral gavage on a small number of animals and found the same effects(data not shown).

Example 4 (Prophetic Example). Synthesis of [¹⁸F]4-Amino-3-(Fluoromethyl)Pyridine

Based on the report by Lee et al on the synthesis of fluoroalkylpyridines with fluorine-18 (Lee et al., 1999), this example depicts apossible synthetic route to generate[¹⁸F]-3-fluoromethyl-4-aminopyridine by protecting the amine of4-aminopyridine-3-methanol with Boc, followed by nucleophilicsubstitution of the benzyl alcohol with ¹⁸F⁻, and boc deprotection.

A solution of Boc₂O (0.20 mol) in CH₂Cl₂ (100 mL, not anhydrous) isadded over 20 min to a stirred suspension of 4-aminopyridine-3-methanol(0.20 mol) in CH₂Cl₂ (200 mL). The resulting solution is stirred at roomtemperature for 25 min (TLC) and acidified with 1 M HCl (230 mL, 0.23mol). The phases are separated, and the aqueous layer is washed withCH₂Cl₂. The combined organic extracts are dried (MgSO₄) and evaporatedin vacuum to give compound 2 which is used in the next step withoutfurther purification.

¹⁸F water is added to the reaction vessel followed by [¹⁸F⁻] K222 (2 mg)in acetonitrile (500 ml), and K₂CO₃ (0.1 mol dm⁻³, 50 ml) and dried at100° C. for 20-30 min. Compound 3 (1 mg) in acetonitrile (1000 mL) isadded. The reaction vessel is sealed and heated at 100° C. for 10 min.The reaction mixture is cooled, washed from the reaction vessel withwater (1.5 mL) and collected in a vial. 3 mL of CF₃COOH are added to thevial and the reaction is heated in a microwave (75 W, 140° C.) for 3min. Subsequently, the reaction mixture is purified by reverse phaseHPLC. Finally, the fractions containing the product are diluted to 5 mLin PBS.

Example 5 (Prophetic Example). Synthesis of [¹⁸F]4-Amino-3-(Fluoroethyl)Pyridine

The synthesis of compound 8 starting from compound 5 may be performedusing the same procedure to Example 4.

Example 6 (Prophetic Example). Synthesis of [¹⁸F]4-Amino-3-Fluoropyridine

This example depicts a possible synthetic route to generate[¹⁸F]-3-fluoro-4-aminopyridine by double deprotonation of N-bocprotected 4-aminopyridine followed by reaction with [¹⁸F]-F2 and Bocdeprotection. Other synthesis including using the recently reportedPd-mediated electrophilic synthesis (Lee et al., 2011) may also beapplicable for synthesizing [¹⁸F]-3-fluoro-4-aminopyridine.

To a solution of 9 (200 mmol) in THE (500 mL) at −78° C. is added t-BuLi(282 mL, 1.7 M, 480 mmol) in pentane over 70 min. The resulting brightyellow suspension is stirred at −78° C. for 20 min and at −15° C. for 2h. Subsequently, [¹⁸F]-F2 is bubbled through 5 ml of solution containinglithiated species 10. After 20 min, 3 mL of CF₃COOH are added to thesolution and the reaction is heated in a microwave (75 W, 140° C.) for 3min to afford compound 11.

Alternatively, the ¹⁸F-labeled versions of 3-F-4-AP and 3-MeF-4-AP aresynthesized as depicted below based on the synthesis of similar PETmarkers (FIG. 8) (Zhou, et al., 2009; Lee, et al., 1999; Dolle, et al.,2005; Cai, et al., 2008; Chun, et al., 2012).

The proposed synthesis of [¹⁸F] 3-F-4-AP uses iodonium salts for highefficiency synthesis of aryl fluorides (Chun, et al., 2012). This tracermay be useful to evaluate lesion size and lesion load in multiplesclerosis patients. This tracer may also be useful in patients withParkinson's disease, stroke, Cerebral palsy, Alzheimer disease, ALS,Lambert Eaton, brain tumors and other diseases.

Synthesis of [¹⁸F] 3-F-4-AP (13): Koser reagent is added to a solutionof 4-[(tert-butoxycarbonyl)amino]pyridin-3-ylboronic acid (10) in CH₂Cl₂and stirred until formation of{4-[(tert-butoxycarbonyl)amino]pyridin-3-yl}(phenyl)iodonium (11). After16 h the solvent is removed under vacuo and the product (11) purifiedusing standard techniques. Next{4-[(tert-butoxycarbonyl)amino]pyridin-3-yl}(phenyl)iodonium (11) isdissolved in DMF and [(crypt-222)K]⁺ ¹⁸F⁻ is added to generate [¹⁸F]N-(3-fluoropyridin-4-yl)carbamate (12). Finally, [¹⁸F]N-(3-fluoropyridin-4-yl)carbamate (12) is treated with aqueous HCl todeprotect the amino group and yield the final product [¹⁸F]3-fluoropyridin-4-amine also known as [¹⁸F] 3-fluoro-4-aminopyridinineor [¹⁸F] 3-F-4-AP

Example 7 (Prophetic Example). Use of the Compounds for Imaging inAnimal Models of Demyelination

Once the radiolabeled markers are obtained, the inventors will performimaging studies in several mouse models of demyelination. The compoundsare tested for imaging demyelination as previously described (Stankoffet al., 2011). The use of different mouse models will enable theinventors to assess whether 4-AP mainly targets potassium channels inneurons or also channels in other cells such as lymphocytes. Suitableanimal models, in particular, mouse models, are contemplated as follows.

DTA Model. The inventors have generated a new mouse model (DTA) ofwidespread CNS demyelination wherein the ablation of oligodendrocytes isaccomplished via cell-specific activation of diphtheria toxin (DT-A)expression in young adult animals (Traka et al., 2010). This approachresults in widespread DT-A-mediated death of mature oligodendrocytes andextensive demyelination throughout the CNS (Traka et al., 2010). At thepeak of disease the DTA mice developed severe tremor and ataxia. Thesemice demonstrate a gradual recovery that culminates in full attenuationof the disease symptoms by approximately 70 dpi, which correlates withthe repopulation of oligodendrocytes and remyelination. This modelprovides widespread and extensive demyelination of the CNS.

Cuprizone-induced demyelination. Feeding of cuprizone (bis-cyclohexanoneoxaldihydrazone) to young adult mice induces a synchronous andconsistent demyelination of the corpus callosum (Matsushima and Morel,2001; Stidworthy et al., 2004). Demyelination and oligodendrocyteapoptosis do not involve T cells or breakdown of the blood-brain barrierin this model and the mice do not display any clinical symptoms.Following the loss of oligodendrocytes and demyelination, there is arepopulation of the oligodendrocytes in the corpus callosum and robustremyelination. The inventors have considerable experience using thecuprizone protocol to examine the demyelination and remyelinationprocesses (Gao et al., 1999; Lin et al., 2004). The cuprizone modelprovides a nice system in which to image highly reproducible, focaldemyelinated lesions that do not involve peripheral immune systeminfiltration.

Experimental autoimmune encephalomyelitis (EAE). EAE which is consideredthe best animal model of MS, can be induced in a variety of species oflaboratory animals by immunization with either myelin or one of itscomponents (Prog. Clin. Biol. Res., 1984; Zamvil and Steinman, 1990;Martin and McFarland, 1995). EAE is an immune-mediated demyelinatingdisease that displays many of the clinical, pathologic, andimmunological features of MS (Behi et al., 2005). Clinical symptomscorrelate with focal inflammatory demyelinated lesions in the spinalcord of the affected animals. The EAE model is capable of providing themost MS-like lesions, which include loss of oligodendrocytes,demyelination and T cell infiltration.

Other animal models of demyelination, such as a lysolecithin injectionmodel, may also be used in the study of demyelination associateddiseases. Animal models other than mouse models may also be used. Itwill be obvious to those skilled in the art to choose an appropriateanimal model to adapt to intended research purposes.

Traumatic brain injury models. In these models a traumatic brain injuryis caused in mice or rats by a controlled impact (using a pendulum or aweight) or by an explosion.

Example 8. (Prophetic Example) Use of the Compounds Described Herein forDiagnosis and Evaluation of MS Progression

A detectable amount of the compound described herein, such as[¹⁸F]-3-fluoromethyl-4-aminopyridine, or [¹⁸F]-3-fluoro-4-aminopyridine,is introduced in the patient body via a pharmaceutically acceptableroute known in the art. The patient is positioned inside a PET scanneror an instrument capable of detecting radiation emitted by the compoundas typically done in the art. The localization of the radioactive traceris done using a computer, which can provide images of the localizationand extent of demyelinated axons.

Example 9. Methods

Non-radioactive Synthesis: Non-radioactive synthesis has been performedusing standard techniques in organic chemistry. Reactions were monitoredby TLC and products characterized by ¹H, ¹³C and ¹⁹F NMR, andhigh-resolution mass spectroscopy.

All chemicals were ordered from Sigma unless otherwise specified. Animalprotocols were approved by IACUC.

Synthesis of tert-butyl N-[3-(hydroxymethyl)pyridin-4-yl]carbamate (8):To a solution of 4-aminopyridine-3-methanol (3) (Alfa Aesar) (806 mg,6.5 mmol) in CH₂Cl₂ (10 mL) a solution of di-tert-butyl-dicarbonate(1.43 g, 6.56 mmol) in CH₂Cl₂ (5 mL) was added and stirred at roomtemperature for 1 h (TLC). After 1 h, the solution was acidified with 1N HCl (7.4 mL, 7.4 mmol). The phases were separated and the aqueousphase was washed with CH₂Cl₂. The aqueous layer was mixed with a freshportion of CH₂Cl₂ (10 mL) and treated with K₂CO₃ (711 mg, 5.1 mmol). Thephases were separated and treated with additional amounts of CH₂Cl₂. Thecombined organic extracts were dried (MgSO₄) and evaporated in vacuo togive a 60:40 mixture containing desired product 8 and the O-linkedcarbonate. Attempts to purify the N-carbamate by flash chromatographyfrom the O-carbonate were unsuccessful due interconversion between thesetwo species in solution at room temperature. ¹H-NMR (CDCl₃, 500 MHz) δ:1.53 (9H, s), 4.67 (2H, s), 4.83 (2H, br s), 7.95 (1H, s), 8.07 (1H, d,J=5.5 Hz), 8.28 (1H, d, J=5.5 Hz), 8.48 (1H, s). This product has beenpreviously synthesized through a different route (Mochizuki, et al.,2011).

Synthesis of tert-butyl N-[3-(fluoromethyl)pyridin-4-yl]carbamate (9):To a solution of triethylamine (450 μL, 2.76 mmol) in CH₂Cl₂ (5 mL) at−78° C. was added XtalFluor-E® (473 mg, 207 mmol) and the product fromthe previous reaction (310 mg, 1.38 mmol in 5 mL CH₂Cl₂). The reactionwas stirred at 0° C. for 15 min (TLC). Subsequently, the reactionmixture was washed with NaHCO₃ (10 mL) and brine (10 mL). The organicphase was dried (MgSO₄) and concentrated in vacuo. The crude product waspurified by flash chromatography to afford 9 (113 mg, 36% yield).R_(f)=0.5 (1:1, hexanes: EtOAc). ¹H-NMR (CDCl₃, 500 MHz) δ ppm: 1.56(9H, s), 5.48 (2H, d, J=48 Hz), 7.12 (1H, br s), 8.12 (1H, d, J=5.5 Hz),8.38 (1H, d, J=3 Hz), 8.32 (1H, s), 8.53 (1H, dd, J₂=5.5 Hz, J₁=1.0 Hz).¹³C-NMR (CDCl₃, 125 MHz) δ: 28.2, 80.6, 82.0 (d, J=15.5 Hz), 113.2,118.0 (d, J=15.5 Hz), 145.6, 150.1, 149.7, 151.7, 152.4. ¹⁹F-NMR (CDCl₃,470 MHz) δ: −209.3 (t, J=48 Hz). HR-MS m/z: 227.1190 (M+H)⁺.

Synthesis of 3-fluoromethyl-4-aminopyridine (5): To a solution of 9 (56mg, 0.25 mmol) in CH₂Cl₂ (3 mL) was added TFA (194 μL, 2.5 mmol) at 0°C. and stirred at room temperature for 5 h (TLC). After 5 h the reactionwas quenched with excess NaOH (1 M). The solvent was evaporated toafford 8 quantitatively. R_(f)=0.2 (MeOH). ¹H-NMR (500 MHz, D₂O) δ: 5.40(2H, d, J=48 Hz), 6.87 (1H, d, J=7 Hz), 7.95 (1H, d, J=7 Hz), 8.09 (1H,s). ¹⁹F-NMR (CDCl₃, 470 MHz) δ: −215.9 (t, J=48 Hz). HR-MS m/z: 127.0666(M+H)⁺.

Synthesis of tert-butyl N-[3-(2-hydroxyethyl)pyridin-4-yl]carbamate(11): Adapted from Spivey et al: To a solution of 4-(Boc-amino)pyridine(1) (2.0 g, 10.3 mmol, 1 eq.) in 25 mL of dry THE at −78° C. was addedt-BuLi (14.5 mL, 1.7 M, 24.7 mmol, 2.4 eq.) in pentane over 30 min. Theresulting bright yellow suspension was stirred at −78° C. for 15 min, at−15° C. for 2 h and then recooled to −78° C. In a separate flask, n-BuLi(7.38 mL, 2.5M, 18.54 mmol, 1.8 eq) in hexanes was added to a solutionof 2-bromoethanol (1.294 mL, 15.45 mmol, 1.5 eq) in 20 mL of dry THE at−78° C. and stirred for 10 min. After 10 min, the bromoethanol solutionwas transferred via cannula to the flask containing lithiatedN-boc-4-aminopyridine over 10 min. The reaction was allowed to warm toroom temperature and the mixture was stirred for 2 h. The reaction wasrecooled to −78° C. and quenched with 5 mL of water. The solution waspartitioned between water (20 mL) and CH₂Cl₂ (30 mL). The phases wereseparated and the extraction was completed with additional portions ofCH₂Cl₂. The combined organic extracts were washed with brine, dried overMgSO₄ and evaporated under vacuum. The crude product was dissolved in asmall amount of CH₂Cl₂ purified by silica gel chromatography (EtOAc) toafford the product 11 (0.678 g, 36% yield). R_(f)=0.15 (EtOAc). ¹H-NMR(500 MHz, CDCl₃) δ: 1.51 (9H, s), 2.81 (2H, t, J=5.0 Hz), 3.80 (1H, brs), 3.94 (2H, t, J=5.0 Hz), 7.92 (1H, d, J=5.5 Hz), 8.15 (1H, s), 8.27(1H, d, J=5.5 Hz), 8.63 (1H, s).

Synthesis of tert-butyl N-[3-(2-fluoroethyl)pyridin-4-yl]carbamate (12):To a solution of Et₃N-3HF (715 μL, 4.39 mmol, 2 eq.) in 5 mL of dryCH₂Cl₂ at 0° C., XtalFluor E® (753 mg, 3.29 mmol, 1.5 eq) was added andstirred for 5 min. After 5 min, 2 (525 mg, 2.195 mmol, 1 eq) was addedand the reaction was monitored by TLC (1:1, hexanes:EtOAc). 15 min laterthe reaction was washed with NaHCO₃ (5 mL) and brine (5 mL), dried withMgSO₄ and the solvent evaporated under vacuum. The crude product wasdissolved in a small amount of CH₂Cl₂ and purified by silica gelchromatography to afford 3 (457 mg, 71% yield). R_(f)=0.4 (1:1,hexanes:EtOAc). Mp=103° C. ¹H-NMR (500 MHz, CDCl₃) δ: 1.53 (9H, s), 2.98(2H, dt, J₂=29 Hz, J₁=5.8 Hz), 4.72 (2H, dt, J₂=47 Hz, J₁=5.8 Hz), 7.04(1H, d, J=7.5 Hz), 7.99 (1H, d, J=5.0 Hz), 8.32 (1H, s), 8.40 (1H, d,J=5.0 Hz). ¹³C-NMR (CDCl₃, 125 MHz) δ: 28.2, 30.4 (d, J=20.1 Hz), 81.6,84.9 (d, J=165 Hz), 113.7, 121.0, 144.7, 149.7, 151.2. ¹⁹F-NMR (CDCl₃,470 MHz) δ: −213.3 (tt, J₂=47 Hz, J₁=29 Hz). HR-MS m/z: 241.1347 (M+H)⁺.

Synthesis of 3-fluoroethyl-4-aminopyridine (6): To a solution of 12 (120mg, 0.5 mmol, 1 eq.) in 5 mL of CH₂Cl₂ was added TFA (191 μL, 2.5 mmol,5 eq) at 0° C. The reaction was allowed to warm up to room temperatureand stirred for 5 h (TLC). After 5 h, the reaction was quenched withexcess NaOH (1 M) and extracted multiple times with CH₂Cl₂. The solventwas evaporated to afford 4 quantitatively. R_(f)=0.5 (MeOH). ¹H-NMR (500MHz, CDCl₃) δ: 2.91 (2H, dt, J₂=26.5 Hz, J₁=6 Hz), 4.26 (2H, br s), 4.69(2H, dt, J₂=47 Hz, J₁=6 Hz), 6.54 (1H, d, J=5.5 Hz), 8.13 (1H, s), 8.15(1H, d, J=5.5 Hz). ¹³C-NMR (CDCl₃, 125 MHz) δ: 30.2 (d, J=20.6 Hz), 84.2(d, J=166 Hz), 110.1, 149.1, 151.0, 151.7. ¹⁹F-NMR (CDCl₃, 470 MHz)−213.3 (tt, J₂=47 Hz, J₁=26.5 Hz). HR-MS m/z: 141.0823 (M+H)⁺.

[¹⁸F] labeling (prophetic): ¹⁸F-labeling will be performed usingcyclotron-generated reagents for nucleophilic and electrophilicfluorination.

Measure Compound Action Potential of demyelinated nerves: 4-AP canenhance the Compound Action Potential in demyelinated nerves. Theeffects of the 4-AP derivatives in the compound action potential ofoptic nerves and/or spinal cords from demyelinated animals will bemeasured according the protocol by Stys et al. Briefly, optic nerveswill be removed postmortem and placed in an oxygenated aCSF solution.Suction electrodes will be used to measure CAP in the presence andabsence of the test compounds (Stys et al., 1991).

Imaging (prophetic): Six mice of each group (DT-A, Cuprizone, EAE andhealthy controls) will be used for the Imaging study. 100 μCi/100 μL of[¹⁸F]-labeled 4-AP derivative will be injected into the tail vein ofanesthesized mice. The imaging sessions will be carried out as 1 hdynamic scan using the MicroPET scanner. The MicroPET data will beprocessed using filter back projection algorithm with attenuation andscatter corrections. In vitro stability studies of the radioactivetracers will be performed according to the protocol by Zhou et al.(2009). Briefly, 2 mL of heparinized mouse blood (C57BL/6N mice) areincubated with the radioactive tracer (˜400 μCi) for 5 min, 30 min, 1 hand 2 h at 37° C. At each time point the blood will be lysed with 3volumes of ethanol and centrifuged. The radioactive species in thesupernatant will be analyzed by radio-TLC and compared to theradioactive tracer's control. In vivo stability: ˜400 μCi of [¹⁸F] 4-APin 200 μL of saline are injected into a mouse by iv injection in thetail vein of an immobilized mouse. Blood samples (0.5 mL) are obtainedvia cardiac puncture under anesthesia at 5 and 30 min post-injection.Afterwards, the plasma is treated with 3 equivalents of acetonitrile andthe pellet separated by centrifugation. The radioactivity species of thesupernatant will be analyzed by radio-TLC and compared to control. Invivo biodistribution time-course study: ˜400 μCi of [¹⁸F] 4-AP in 200 μLof saline will be injected into a mouse by iv injection. At 5 min, 30min, 1 h and 2 h post injection the mice will be sacrificed and bloodtissues and organs removed, weighed and counted using a Beckman counterwith standard diluted aliquots of the sample. The percent injected doseper gram of tissue will be calculated.

Neurological evaluation (prophetic): the effects of the 4-AP derivativesin demyelinated animals will be evaluated using a rotorod to test forbalance and coordination, the inventors will also measure changes intremor and other functions.

Pharmacology of 4-AP derivatives (prophetic): metabolic stability,membrane permeability, toxicity, pharmacokinetic and drug distributionstudies will be conducted with the assistance of a third party researchcontract organization.

Measurement of 4-AP and derivatives distribution in mice (prophetic):The distribution of 4-AP and derivatives will be measured usingMALDI-IMS, whole body autoradiography, or organ autoradiography.

Example 10. Blockage of K⁺ Channels by 4-AP Derivatives Using VoltageClamp

The inventors tested the ability of compounds 1-7 to block voltage-gatedK⁺ channels expressed in Xenopus oocytes using the cut-open voltageclamp technique described by Stefani and Bezanilla. For this experiment,Shaker K⁺ channel from D. megalonaster was chosen as the archetypicalvoltage gated K⁺ channel that gives name to the family. Shaker shares anidentity ranging from 69%-79% with neuronal K_(v)1.1, K_(v)1.2,K_(v)1.3, K_(v)1.4, K_(v)1.6 that are among the presumed targets of 4-APand its sensitivity to 4-AP is comparable to other K_(v)1 channels(Gutman, et al., 2005; McCormack, et al., 1994). In order to compare therelative potency of the different 4-AP derivatives, each drug wasapplied at increasing concentrations and the ratio between the K+current with and without drug was computed (FIG. 5).

Electrophysiology: Electrophysiology studies are conducted according tothe protocol by Stefani and Bezanilla (1998). Briefly, K⁺ channel ShakercRNA is injected into Xenopus oocytes 24 h after their surgicalextraction from adult frogs. 1-5 days after injection channel currentsare recorded using the cut-open voltage-clamp. Each molecule is added tothe external solution at a range of concentrations and K+ currentsrecorded and compared to those with 4-AP.

Expression of Shaker K+ channel in Xenopus laevis oocytes: K⁺ channelexpression in Xenopus oocytes membranes was achieved by injectingapproximately 50 ng of WT Shaker cRNA (kit Ambion) into the oocytes 24 hafter surgical extraction from adult frogs and collagenase treatment.Injected oocytes were maintained in a standard oocytes solution (100 mMNaCl, 5 mM KCl, 2 mM CaCl₂), and 10 mM Hepes at pH 7.5) at 16.5° C. andrecordings were performed 1-3 days after injection.

Recording of K⁺ currents in Xenopus oocytes: K⁺ currents were recordedfrom oocytes expressing Shaker K⁺ channels using the cut-open voltageclamp technique as described by Stefani and Bezanilla. The internalsolution was 120 mM KOH, 20 mM HEPES-methyl sulfonate (MES) pH 7.4, 2 mMEGTA. The external solution was 12 mM KOH, 105 mMN-methyl-D-glucamine-MES pH 7.4, 20 mM HEPES, 2 mM CaOH. To assess theeffects of the 4-AP derivatives, the drug under study was added inincremental concentrations by exchanging the external solution (top andguard chambers) several times. After application, cells werevoltage-clamped at least 5 min at 0 mV, then voltage-clamped at −80 mVfor 1-2 min. K⁺ currents were generated by applying series of 50 mspulses from −70 mV to +40 mV in increments of 10 mV. The effect of thedrug was assessed by measuring the relative intensity of the K⁺ currentbefore and after applying varying drug concentration at a constantvoltage (typically +20 mV) and at the end of the test-pulse. Analysis ofthe traces was done using an in-house software. The half-maximalinhibitory concentration (IC₅₀) for each drug was calculated by plottingthe relative K⁺ current vs. concentration and fitted to the Hillequation using the software Origin.

In this experiment, it was demonstrated that 4-AP and 3-F-4AP are themost potent compounds with half-maximal inhibitory concentrations (IC₅₀)around 0.27 mM (4-AP: IC₅₀=0.29 mM, 95% C.I.=0.21-0.41 mM; 3-F-4-AP:IC₅₀=0.25 mM, 95% C.I.=0.13-0.44 mM), which is similar to what has beenreported for other Shaker-like channels (Gutman, et al., 2005). AlthoughBerger et al described 3-F-4-AP as being less potent than 4-AP ineliciting muscle twitches in dissected mouse hemidiaphragms, it wasfound to have comparable potency in blocking Shaker K⁺ channel. In thisassay, 3-MeOH-4-AP and 3-MeF-4-AP were found to be between 15 and 25times less potent than 4-AP (3-MeOH-4-AP: IC₅₀=4.38 mM, 95% C.I.=3.4-5.6mM; 3-MeF-4-AP: IC₅₀=7.45 mM, 95% C.I.=6.2-9.0 mM). In contrast,3-EtF-4-AP and 2-F-4-AP have IC₅₀ values greater than 10 mM (95% C.I.not determined). These results demonstrate that only small modificationsin the 2 position of 4-AP are permitted (e.g. 3-F-4-AP, 3-MeF-4-AP,3-MeOH-4-AP) whereas larger modifications such as 3-EtF-4-AP orsubstitution in the 2 position such as 2-F-4-AP significantly diminishactivity. In this experiment, it was also observed that these drugs aredifficult to wash, out which is similar to what has been reported for4-AP (McCormack, et al., 1994), suggesting a similar mode of binding.

Example 11. Effects of 4-AP Derivatives on the Compound Action Potentialof Dissected Optic Nerve

It is known that 4-AP can significantly enhance action potential ofdemyelinated fibers (Sherratt, et al., 1980; Devaux, et al., 2002). Inorder to determine if the drugs could also be effective in enhancingcompound action potentials (CAP), the effects of these compounds on theCAP of hypomyelinated optic nerves from Shiverer mice (shi^(−/−)) andcontrol mice (shi^(+/−), shi^(+/+)) were tested. Shiverer mice lackcompact myelin in the CNS due to a null mutation of the myelin basicprotein gene (MBP). The results of this experiment are shown in FIG. 5.

Dissection of optic nerves from Shiverer mice: optic nerves weredissected from 12-16 week old Shiverer (shi^(−/−)) and control mice(shi^(+/−) and shi^(+/+)). Mice were euthanized by CO₂ overdose and theoptic nerves were quickly dissected between the eyeball and the opticchiasm. The nerves were incubated for 30 min at 37° C. in oxygenated(95% O₂, 5% CO₂) aCSF solution (126 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 26 mMNaHCO₃, 2 mMCaCl₂, 10 mM dextrose, pH 7.5) before the experiment.

Optic nerve electrophysiology: compound action potentials (CAP) fromhypomyelinated nerves (Shiverer mice, shi^(−/−)) and myelinated nerves(litermate controls, shi^(+/−) and shi^(+/+)) were recorded usingsuction electrodes as described by Stys et al. Briefly, the dissectedoptic nerve was placed inside a chamber containing oxygenated (5% CO₂,95% O₂) aCSF (300 μL) between two suction electrodes (stimulus andrecording electrodes) forming a tight seal on each end. Two additionalelectrodes were placed in the bath for reference. A supramaximal pulse(250 mV, 20 μs) was applied at the stimulating end of the nerve. Theresulting CAP was amplified from the recording electrode using a highimpedance low-noise amplifier (EG&G Princeton Applied ResearchCorporation) and filtered and sampled at 10-100 kHz. To assess theeffects of the 4-AP derivatives on the CAP, the drug under study wasadded in incremental concentrations to the recording chamber after theCAP was allowed to stabilize for 5 min while pulsing repeatedly. Aftereach measurement the chamber was washed for 5 min (flow 1 mL/min) withoxygenated aCSF. The study was conducted at 22.2±1.3° C. to allow forslower conduction and the temperature was monitored throughout theexperiment. CAP recordings were acquired with a SBC6711 board(Innovative Integration) controlled by in-house written software.Analysis of the traces was done using an in-house software. Thehalf-maximal effective concentration (EC₅₀) for each drug was calculatedby plotting the final over initial amplitude vs. concentration andfitted to the Hill equation using Origin.

This experiment shows the typical differences between normallymyelinated nerves and hypomyelinated nerves. The Shiverer'shypomyelinated nerves conducted much slower (average conduction velocity0.59±0.10 m/s vs. 1.4±0.3 m/s at 22° C.), had a smaller CAP amplitude(20-30% compared to myelinated nerves) and showed a larger undershootthan control nerves. Addition of 4-AP to normally myelinated nervescaused small increases in CAP amplitude of around 5% and broadening ofthe signal, whereas addition of 4-AP to hypomyelinated nerves causedlarge increases in CAP amplitude of 2-4 fold generating a CAP that,although delayed, almost looked like a normally myelinated nerve (FIG.4A). As for the effect of the different derivatives, 4-AP and 3-F-4-APwere found to be the most potent in enhancing the CAP with half-maximaleffective concentrations (EC₅₀) of 59.2 μM (95% C.I. 43-81 μM) and 96 μM(95% C.I. 29-323 μM) respectively. The derivatives 3-MeOH-4-AP and3-MeF-4AP were around 4-6 times less potent with EC₅₀'s around 390 μM(3-MeOH-4-AP: IC₅₀=386 μM, 95% C.I.=295-505 μM; 3-MeF-4-AP: IC₅₀=286 μM,95% C.I.=234-648 μM). As expected, 2-F-4AP had no effect demonstratingthat the observed effects with the other derivatives are specific.3-EtF-4-AP was not included in this experiment since it was alreadyfound to be inactive by voltage clamp. The trend observed in thisexperiment was consistent with what was observed in the voltage-clampexperiment indicating that the increase in CAP is due to blockage ofvoltage-gated K⁺ channels. Interestingly, the EC₅₀ values calculatedfrom this experiment were significantly lower than what was measuredusing voltage-clamp.

Example 12. Pharmacology of 3-F-4-AP and 4-AP

The permeability of 3-F-4-AP and 4-AP to an artificial membrane made ofporcine brain polar lipids was tested. In this experiment, the inventorsincluded highly permeable verapamil and lowly permeable theophylline ascontrols. The inventors found 3-F-4-AP to be 6.6-times more permeablethan 4-AP (P_(e): 15.6±0.6 nm/s vs. 2.36±0.03 nm/s, FIG. 6A). This valuecorrelates well with the predicted partition coefficients inoctanol/water for these drugs (c Log P: 0.26 vs. 0.03, Pearson r=0.997,P value=0.0033).

The stability of these drugs in mouse plasma and mouse liver microsomeswas also tested. Liver microsomes contain large amounts of cytochromeP450 and can be used to estimate the metabolic stability of drugs. Inthis experiment, highly stable verapamil and lowly stable propanololwere included as controls. Both drugs were found to be stable in plasma(>93% remaining after 1 h) and 3-F-4-AP was found to be 2.7-times morestable than 4-AP in microsomes (4-AP: t_(1/2)=53±10 min; 3-F-4-AP:t_(1/2)=144±11 min) (FIG. 6A).

Pharmacokinetic profiling of 4-AP and 3-F-4-AP in mice after a singleintravenous dose of the drugs at 0.75 mg/kg (FIG. 6B) was alsoperformed. In this experiment, it was found that 4-AP and 3-F-4-AP havea short half-life in plasma (0.33 h and 0.34 h, respectively) and amoderate half-life in brain (1.9 h and 1.43 h, respectively).Interestingly, it was observed that 3-F-4-AP reaches a significantlyhigher concentration in the brain indicated by the ratio between themaximum dose in the brain over the maximum dose in plasma(C_(brain)/C_(plasma): 0.214±0.17 vs. 0.10±0.05). This result isconsistent with the previous experiment which showed that 3-F-4-AP candiffuse faster across hydrophobic membranes. Taken together theseexperiments demonstrate that 3-F-4-AP has better stability and betterbrain permeability than 4-AP.

In vivo effects: 10-week-old female C57Bl/6J mice were given anintraperitoneal injection of the drug under investigation and monitoredcontinuously for 4 h. After 4 h no signs of drug effects could beobserved. At least 72 h passed between injections to the same mice.

Parallel artificial membrane permeability assay (PAMPA): Permeabilitystudies were performed as previously described by Sugano, et al., 2011.A 96-well microplate (acceptor compartment) was filled with PBScontaining 5% DMSO. A hydrophobic filter plate (donor compartment) wasplaced atop the buffer-filled plate and the filter surface wasimpregnated with 5 μL solution of porcine polar brain lipids (AvantiLipids) in dodecane (1% w/v). 150 μL of the test compounds dissolved inPBS containing 5% DMSO (compound concentration 0.5 mM) was added to thedonor compartment and covered. The only barrier between the twocompartments was the artificial BBB membrane containing the porcinepolar brain lipids. The whole system was incubated for several hours.Time of incubation was chosen considering c Log P of tested compounds (4h for Verapamil and 16 h for 4-aminopyridine, 3-fluoro-4-aminopyridineand theophylline). Samples from the acceptor compartment were analyzedby UV-VIS spectrophotometry (4-aminopyridine: 260 nm,3-fluoro-4-aminopyridine: 265 nm) and compared to reference solutions.

Stability in plasma: Plasma stability was conducted by incubating eachcompound at initial concentration of 1 μM in mouse plasma for 60minutes. Samples were collected at 0, 20, 40 and 60 minutes and thereaction was stopped by addition of 1 vol. of acetonitrile. The loss ofcompound was determined using LC-MS comparing the peak area at severaltime points. Half-life time was calculated from linear regression oftime course data.

Stability in microsomes: study compounds were incubated at initialconcentration of 1 μM with liver microsomes from CD1 mouse (0.04 mg/mL)in PBS in the presence or absence of enzyme cofactors (1.3 mM NADP⁺, 3.3mM MgCl₂, 3.3 mM G6P and 1 U/ml G6PDH, 1 mM UDPGA and 4.7 μg/mlAlamethicin). After t=0, 20, 40 and 60 min, a sample was removed and thereaction was stopped by adding 1 volume of acetonitrile. The loss ofcompound was determined by LC-MS analysis comparing the amount ofcompound in the sample to the respective reference samples (withoutcofactors). To ensure that the assay is reliable, Propanolol andVerapamil were included as control compounds. The results werenormalized for reaction volume and protein concentration.

LC-MS: The following conditions were used for LC-MS analysis. Solvent A:Water (0.1% Formic acid). Solvent B: Acetonitrile (0.1% Formic acid).Flow rate: 0.5 ml/min. Gradient conditions: 0.0-0.5 min 95% B, 0.5-6.0min 5% B, 6.0-6.5 min 5% B, 6.5-6.6 min 95% B, 6.6-7.5 min 95% B.Running time: 7.5 min. Injection volume: 40 μl. Column: Luna HILIC,150×4.6 mm, 3 m. Ionization mode: ESI positive. MS mode: MultipleReaction Monitoring (MRM). Capillary voltage: 4500 V. Nebuliser gas: 40psi. Dry gas: 9 μL/min. Dry Temperature: 300° C. HPLC and MS/MSparameters: 4-aminopyridine retention time 3.5 min, ion product 94.9.3-fluoro-4-aminopyridine retention time 4.0 min, ion product=112.9.

Pharmacokinetic study: 39 mice (CD-1, 6-weeks old, female) were used inthe study. 18 mice were administered 4-aminopyridine, 18 mice wereadministered 3-fluoro-4-aminopyridine and 3 were left untreated. Thedrugs were dissolved in PBS and administered via tail-vein injection toachieve a dose of 0.75 mg/kg of body weight. At specific times postinjection (10 min, 30 min, 1 h, 2 h, 4 h and 24 h) blood and brainsamples were collected. Blood samples were transferred into tubescontaining 5% EDTA, stored on ice, and centrifuged (4° C., 1000 rpm, 15min). Plasma (upper phase) was transferred to a new tube and stored at−80° C. for further analysis. Brain tissue samples were collected afterintracardial perfusion of the mouse. Brain tissue samples were stored at−80° C. for further analysis. Drug quantification in plasma: Briefly, toa 1.5 mL Eppendorf tube containing 50 μl of plasma, 200 μl of ice-coldacetonitrile containing 1,000 ng/ml Progesterone (used as internalstandard) was added in order to precipitate the proteins. The sample wasvortexed, mixed and centrifuged at 4000×g for 10 min at 4° C. to removeprecipitates. 140 supernatant was collected and transferred to a 500 μl96-wel polypropylene plate and covered using silicone plate mat. 40 μlof sample was injected in into LC-MS. Using the same sample preparationprocedure 11 standard solutions ranging from 50 ng/mL to 100 μg/mL wereprepared, analyzed by LC-MS and used as a calibration curve to correlatepeak area of the samples to concentration. Drug quantification in brain:the mouse brain was weighted and 1 mL of water per 400 mg of tissue wasadded. The sample was homogenized using an electric tissue homogenizer.To a 1.5 mL Eppendorf tube containing 50 μl of homogenized sample, 200μl of ice-cold acetonitrile containing 1000 ng/ml Demeclocycline (usedas internal standard) was added in order to precipitate the proteins.The sample was vortexed, mixed and centrifuged at 4000×g for 10 min at4° C. to remove precipitates. 140 μl supernatant was collected andtransferred to a 500 μl 96-well polypropylene plate and covered usingsilicone plate mat. 40 μl of each sample was injected into LC-MS. Usingthe same sample preparation procedure 11 standard solutions ranging from50 ng/mL to 100 μg/mL were prepared, analyzed by LC-MS and used as acalibration curve to correlate peak area of the samples toconcentration.

Data analysis: The Hill equation used to fit the data from thevoltage-clamp and the optic nerve experiments was as follows:y=y₀+(y_(f)−y₀)*x^(n)/(k^(n)+x^(n)); where n refers to the Hillcoefficient (typically 1±0.1) and k refers to EC₅₀ or IC₅₀. y₀ and y_(f)refers to the origin and final ordinate values and were fixed at 1±0.1or 0±0.1 depending on the experiment. EC₅₀=10^(<log EC50>); where <logEC₅₀>=average of log EC₅₀ values from all experiments with the samedrug. 95% C.I.=[Upper Limit . . . Lower Limit]; where U.L.=₁₀^((log EC50+s.d.)) and L.L.=10^((log EC50−s.d.)). The half-life(t_(1/2)) in the stability and pharmacokinetic experiments wascalculated by fitting the data to the equation C_(t)=C₀*exp(−k*t) whereC₀=initial concentration, C_(t)=concentration at time t, and k=ln2/t_(1/2). Student t-test was used to compare results and P<0.05 wasconsidered significant.

Example 13. Evaluation 4-AP Distribution in Partially DemyelinatedBrains Using Autoradiography

The inventors conducted an experiment to evaluate if 4-AP selectivelylocalizes in demyelinated (injured) areas. In this experiment, tritiumlabeled 4-aminopyridine ([³H] 4-AP) was injected into mice containingdemyelinating lesions in the brain. The lesions were caused by priorinjection of lysophosphatidylcholine (LPC), also called lysolecithin,into their brains, which causes focal demyelination at the site ofinjection. Two days after LPC injection, at the peak of demyelination,the animals were injected with [³H] 4-AP (0.4 μCi/g) via tail veininjection. Twenty to sixty minutes after injection of [³H] 4-AP the micewere euthanized and their brains were dissected and frozen. The frozenbrains were then cut into 20 m sections using a cryostat and thesections mounted in slides. The slides were then exposed to radiationsensitive X-ray film at −80° C. in the dark for forty days to capturethe distribution of radioactivity coming from [³H] 4-AP throughout thebrain.

After autoradiographic exposure, the slides were stained for myelinbasic protein (MBP) using immunohistochemistry and imaged usingfluorescent microscopy to determine the areas of demyelination. Theresults of this experiment are shown in FIGS. 7A-E.

In this experiment, partial demyelination in distinct areas on the rightside of the corpus callosum where lysolecithin was injected (FIG. 7C,circled in red) were seen. In those areas, the autoradiographic signalappears darker than the rest of the corpus callosum (FIG. 7D). Theinventors quantified the signal in those areas and observed astatistically significant increase in signal in demyelinated areas (FIG.7E).

This experiment demonstrates that 4-AP selectively localizes to greymatter areas and there's virtually no 4-AP in white matter areas. It wasalso observed that demyelination of white matter areas causes a localincrease in the autoradiographic signal indicating that 4-AP localizesto demyelinated areas but not myelinated areas. The conclusion is that4-AP does not bind to white matter areas unless there is demyelination.

It was shown that fluorinated 4-APs can block Shaker K⁺ channel similarto 4-AP, that fluorinated 4-APs can enhance compound action potential ofdysmyelinated optic nerves but have very little effect on normallymyelinated optic nerves, and that fluorinated 4-APs have very similar invivo effects as 4-AP. It was also shown that fluorinated 4-APs haveenhanced permeability to the CNS relative to 4-AP. As 4-AP localizes todemyelinated areas and fluorinated 4-APs have very similar biologicalactivity to 4-AP, it can be inferred that fluorinated 4-APs alsolocalizes to demyelinated lesions. As fluorinated molecules can be usedas PET tracers simply by exchanging the natural isotope of fluorine(¹⁹F) for the positron emitting isotope ¹⁸F and this exchange does notalter the biological properties of the molecule, the evidence supportsan inference that ¹⁸F-labeled 4-APs can serve as PET tracers fordemyelination.

Example 14 (Prophetic). Distribution of [¹⁴C] 3-F-4-AP in PartiallyDemyelinated Rat Brain and Spinal Cord Containing Demyelinated LesionsUsing Autoradiography

Rats will be injected with LPC in the brain and spinal cord to createfocal demyelinated areas at the sites of injection. 1-6 days after LPCinjection, the rats will be injected intravenously with [¹⁴C] 3-F-4-AP(0.5 mg/kg, 0.5 uCi/g). 20-90 min after [¹⁴C] 3-F-4-AP injection, therats will be euthanized and their brains removed. Thin sections of thebrain will be prepared using a cryostat and mounted into glass slides.The slides will be then exposed to a radiation sensitive film for up to6 weeks. After exposure, the film will be developed and the slides willbe processed for IHC. The distribution of the drug on the brain revealedby autoradiographic signal and compared with the distribution of myelinrevealed by IHC. In some experiments, other ¹⁴C labeled fluorinatedderivatives of 4-AP are used. In some experiments, ³H labeledfluorinated derivatives of 4-AP are used. In some embodiments, differentrodent models of demyelination are used. In some embodiments, differentspecies may be used.

Example 15 (Prophetic). Distribution of [¹⁸F] 3-F-4-AP in PartiallyDemyelinated Rat Brain and Spinal Cord Using Pet Scanner

The inventors will inject 0.005-50 mCi of [¹⁸F] 3-F-4-AP or other¹⁸F-labeled 4-AP derivative into LPC treated rats. Immediately afterinjection of the tracer, dynamic emission scan will be performed in 3Dacquisition mode on the animal using a GMI microPET/SPECT/CT system. Inorder to quantify the images, the signal will be integrated in thelesion and compare it to the signal in the same area in a controlanimal. The results will be analyzed using statistical tests. In someexperiments, other ¹¹C labeled fluorinated derivatives of 4-AP are used.In some embodiments, different rodent models of demyelination are used.In some embodiments, different species may be used. In some embodiments,the species will be humans.

Example 16. (Prophetic) 4-AP Preferentially Localizes to TBI LesionsUsing Autoradiography

4-AP has a higher uptake in demyelinated (injured) white matter areasthan in the rest of the white matter. In order to determine if 4-APlocalizes to areas of the brain affected by a traumatic brain injury,the inventors will conduct a similar experiment in a rat model of TBI(FIG. 9A). The inventors will induce traumatic brain injuries in ratsusing the controlled impact model. Afterwards, the animals will beinjected with ¹⁴C-labeled 4-AP (0.1-0.5 μCi/g). The animals will beeuthanized 30 to 90 min later and their brains removed. Thin sections oftheir brains will be prepared and the localization of the drugdetermined using autoradiography (FIG. 9B). Subsequently the lesionedareas will be examined by immunohistochemistry to identify the traumaticinjured areas. The autoradiographic signal corresponding to thoseregions will be quantified and the results evaluated using analysis ofvariance (ANOVA).

We also plan to perform a similar experiment using [¹⁴C]2-deoxy-d-glucose (2-DG). 2-DG has a similar distribution pattern in thebrain (higher uptake in grey matter areas than white matter areas).However, 2-DG it is not expected to accumulate in injured areas of thebrain to a significant degree. This experiment will serve as a controland provide a preview of the comparison of [¹⁸F]-labeled 4-AP with [¹⁸F]FDG using PET.

Example 17 (Prophetic)—Compare the Distribution of 3-F-4-AP and3-MeF-4-AP in the Brain of Animals with Demyelinating Lesions

Using the same autoradiographic technique we will evaluate the braindistribution of [¹⁴C] 3-F-4-AP and [¹⁴C] 3-MeF-4-AP. These will be themolecules used for PET imaging. Both of these molecules have similaraffinity to K+ channels as 4-AP and better brain permeability. Thisexperiment will allow us to identify the best candidate for imaging. Inaddition, we will include [¹⁴C] 2-F-4-AP which has the same molecularweight as [¹⁴C] 3-F-4-AP and very similar brain permeability but doesnot bind to K⁺ channels to control for non-specific localization of thistype of molecules.

Similar as with 4-AP, we will perform a dose-response and preblockingexperiments in control rats to determine the best conditions forimaging. Once we optimize the conditions we will perform the experimentsin lysolecithin-injected rats. Based on our statistical calculation, weestimate that in this experiment we will need around 6 rats per group.Similar as before, the results will be analyzed using ANOVA.

Example 18 (Prophetic)—Synthetic Methodology for [¹⁸F] 3-F-4-AP and[¹⁸F] 3-MeF-4-AP

Fluorine-18 is the most appropriate radionuclide for PET as its lowpositron energy allows for sharper resolution, and its longer half-life(109.8 min vs. 20 min for ¹¹C) allows for off-site production andcommercialization. In order to be able to use fluorinated 4-APs as PETtracers, a quick radiolabeling strategy will be necessary (¹⁸Fhalf-life: 109 min). See FIG. 8.

The proposed synthesis of [¹⁸F] 3-F-4-AP makes use of the recentlydeveloped method of using iodonium salts for high efficiency synthesisof aryl fluorides (Chun, et al., 2012). In comparison, [¹⁸F] 3-MeF-4-APpossesses an aliphatic fluoride that it is expected to be facile tosynthesize.

Example 19 (Prophetic)—Pet Imaging in Small Animal Models of TBI

Immediately after synthesizing the ¹⁸F-labeled compounds, a dynamicemission scan will be performed in 3D acquisition mode in the TBIinduced animals using a GMI microPET/SPECT/CT system. The resolution ofthe microPET scanner is limited to ˜1 mm. In addition, a post-mortemautoradiography will be conducted after the scan to further verify thelocalization of the tracer.

All of the methods and apparatuses disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand apparatuses and in the steps or in the sequence of steps of themethods described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-15. (canceled)
 16. A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R₁, R₂, R₃, andR₄ are each a functional group independently selected from the groupconsisting of: H, X, (CH₂)_(n)X, CH₂OCH₂CH₂X, NH₂, CH₂OH, CX₃, CFX₂,CF₂X, OCH₃, OCH₂X, OCHX₂, OCHFX, or OCX₃, OCFX₂, and OCF₂X; R₅ is afunctional group selected from the group consisting of: H, (CH₂)_(m)X,OH, COOCX₃, COOCFX₂, COOCF₂X, COOC(CH₃)₃, and COO(CH₂)_(m)X; whereinn=1, 2, 3, 4, or 5; wherein m=2, 3, 4, or 5; wherein X representsfluorine or ¹⁸F; and wherein the compound contains at least one ¹⁸F. 17.The compound of claim 16, wherein at least one hydrogen atom is adeuterium isotope.
 18. The compound of claim 16, wherein the compound is[¹⁸F]3-fluoro-4-aminopyridine.
 19. The compound of claim 16, wherein thecompound 3-[¹⁸F]fluoromethyl-4-aminopyridine.
 20. The compound of claim16, wherein the compound 3-[¹⁸F]fluoroethyl-4-aminopyridine.
 21. Thecompound of claim 16, wherein the compound3-[¹⁸F]trifluoromethyl-4-aminopyridine.
 22. A pharmaceutical compositioncomprising the compound of claim 16 and a pharmaceutically acceptablecarrier.